WO2023125562A1 - Method for detecting multiple target nucleic acids - Google Patents

Abstract

The present invention relates to the field of molecular biology, in particular to a method for detecting multiple target nucleic acids in a single sample container. The present invention particularly relates to a method for multiplex detection based on digital PCR. The method comprises: mixing a primer probe composition, a sample to be tested and an amplification reagent to obtain a reaction system; placing the reaction system in a condition that enables a nucleic acid polymerase to perform hybridization and extension reaction, so as to obtain a reaction product; and performing signal acquisition on the reaction product. The described method can realize detection of multiple target nucleic acids, is simple to operate and can save time.

Description

A variety of target nucleic acid detection methods

Cross References to Related Applications

This application claims the priority of the Chinese patent application CN 202111617233.2 entitled "A Primer Probe for Detection, Primer Probe Set and Its Application" filed on December 27, 2021. The entire content of the application is passed incorporated herein by reference.

technical field

The present invention relates to the field of molecular biology, in particular to a method for the detection of multiple target nucleic acids in a single sample container.

Background technique

The polymerase chain reaction (PCR) is a molecular biology technique for the enzymatic replication of DNA without the use of living organisms. PCR is commonly used in medical and biological research laboratories to undertake a variety of tasks, such as diagnosis of infectious diseases, gene cloning, phenotyping of experimental animals, transcriptome research, detection of genetic diseases, identification of genetic fingerprints, paternity testing, etc. Due to its unparalleled ability to replicate and be precise, PCR is considered by molecular biologists to be the method of choice for nucleic acid detection. In the late 1990s, the real-time fluorescent quantitative PCR (Real Time Quantitative PCR, qPCR) technology and related products launched by the American ABI company developed PCR into a highly sensitive, highly specific and accurate quantitative nucleic acid sequence analysis technology.

Digital PCR (digital PCR, dPCR) technology is an absolute quantitative technology for nucleic acid molecules. It uses the principle of limiting dilution to distribute a fluorescent quantitative PCR reaction system into thousands of individual nanoliter microreactors, so that each The microreactor contains or does not contain one or more copies of the target nucleic acid molecule (DNA target), and then performs single-molecule template PCR amplification at the same time. Different from the fluorescence quantitative PCR method that collects fluorescence during each amplification cycle, digital PCR collects the fluorescence signal of each reaction unit independently after the amplification, and finally uses the principle of Poisson distribution and positive/negative reactions The ratio of cells yields the native copy number or concentration of the target molecule.

Compared with fluorescent quantitative PCR, digital PCR can perform accurate absolute quantitative detection without relying on Ct values and standard curves, and has the advantages of high sensitivity and accuracy. Since digital PCR only judges the two amplification states of "presence/absence" when interpreting results, there is no need to detect the intersection point of the fluorescent signal and the set threshold line, and it does not depend on the identification of the Ct value at all, so the digital PCR reaction and Results Interpretation is greatly reduced by the influence of amplification efficiency, and the tolerance to PCR reaction inhibitors is greatly improved. In addition, the process of distributing the reaction system in digital PCR experiments can greatly reduce the concentration of background sequences that compete with target sequences locally. Therefore, digital PCR is particularly suitable for detecting rare mutations in complex backgrounds. Currently, it is mostly used in In liquid biopsy, the detection of rare mutation markers in the peripheral blood of tumor patients is realized.

In order to realize the multiple detection of digital PCR, a common method is to design a probe for each target, and realize the detection of different targets through the different fluorescence of different probes. However, there are too many types of probes, not only the cost is high, but also the fluorescence background is high, which reduces the sensitivity of detection.

Contents of the invention

In order to solve the above problems, the present invention provides a method for detecting multiple target nucleic acids, including the following:

Mixing the primer probe composition, the sample to be tested and the amplification reagent to obtain a reaction system; the amplification reagent includes nucleic acid polymerase and dNTPs;

placing the reaction system under conditions that allow nucleic acid polymerase to perform hybridization and extension reactions to obtain reaction products;

Place the reaction product at n different signal collection temperatures for n times of signal collection, and perform one signal collection for each signal collection temperature; each signal collection includes at least one signal channel collection;

Analyzing whether there is a difference in the signals acquired by two adjacent signal channels under the same signal channel, and determining the presence or absence of the target nucleic acid in the sample to be tested; and/or,

Analyzing the presence or absence of the signal acquired by the signal channel with the highest signal acquisition temperature under the same signal channel to determine the presence or absence of the target nucleic acid in the sample to be tested;

Wherein, the n is an integer ≥ 2, and n ≤ the number of types of target nucleic acids.

Using the above detection method, the detection of multiple target nucleic acids can be realized (at least 8 multiplex reactions can be realized on digital PCR). The realization of multiple detection does not only depend on the fluorescent channel, but also uses different melting temperatures in the same fluorescent channel (that is, the same signal channel), and can distinguish between different targets by taking a limited number of photos to distinguish the presence or absence of fluorescence. The fluorescent recognition of digital PCR has low requirements, simple operation and time saving.

In some embodiments, one signal acquisition is performed for each signal acquisition temperature; if n different signal acquisition temperatures are used, n times of signal acquisition are performed. In some embodiments, n is the number of signal collections; n≤the number of target nucleic acid types, that is, the number of signal collections≤the number of target nucleic acid types.

In some embodiments, under the same signal channel, the number of target nucleic acid species contained in the signals acquired by two adjacent signal channels differs by at most one; in some embodiments, under the same signal channel, The number of target nucleic acid species contained in the signals collected by two adjacent signal channels differs by one; in some embodiments, the signal collected by the signal channel with the highest signal collection temperature contains at most one target nucleic acid. nucleic acid. In some embodiments, the "one type of target nucleic acid" also includes Type 1 target nucleic acid that is not classified.

In some embodiments, "signal acquisition" includes the following operations: the first signal acquisition is performed on the reaction product at the first temperature, and then the temperature is raised to the second temperature for the second signal acquisition, and then the temperature is continued to the third temperature. The third signal collection is carried out at the temperature, and then the heating and signal collection are continued until all the target nucleic acids in the sample to be tested are detected. It can also include the operation of signal acquisition in continuous cooling, for example, the first signal acquisition is performed on the reaction product at the first temperature, then the temperature is lowered to the second temperature for the second signal acquisition, and then the temperature is continued to be lowered to the third temperature. The signal is collected for the third time at the temperature, and then the cooling and signal collection are continued until all the target nucleic acids in the sample to be tested are detected.

In some embodiments, there is only one type of probe in the primer-probe composition (one type of probe refers to a type of modified detection label or fluorescent group that is the same, and its base sequence can be the same or different. probe), each signal acquisition includes only one signal channel acquisition, that is, each signal acquisition is one signal acquisition under a certain signal channel (fluorescence channel). At this time, two adjacent signal channel acquisitions are two adjacent signal acquisitions, and the number of target nucleic acid species contained in the signals obtained by adjacent two signal channel acquisitions differs by 1, then the number of times n of signal acquisition is equal to the number of target nucleic acid species number of species. For example, the sample to be tested contains 3 kinds of target nucleic acids. Under a certain fluorescence channel, the reaction product is collected at the first temperature for the first signal acquisition (equivalent to signal channel acquisition), and the obtained signal contains 3 kinds of targets. Then the temperature was raised to the second temperature for the second signal collection, and the obtained signal contained 2 kinds of targets, and then the temperature was continued to be raised to the third temperature for the third signal collection, and the obtained signal contained 1 kind of target. The presence of the target nucleic acid can be determined by comparing the signal conditions of two adjacent signal collections. For example, compared to the first signal acquisition, some signals disappear during the second signal acquisition, and the disappeared signal is the first target; compared with the second signal acquisition, another part of the signal disappears during the third signal acquisition. When the signal disappears, another part of the signal that disappears is the second target, and the signal that still exists in the third signal acquisition is the third target. In this way, the existence of all three targets can be determined. In addition, the amount of each target can be determined according to the amount of signal disappearance/presence.

In some embodiments, there are 2 kinds of probes in the primer-probe composition (2 kinds of probes, referring to the modified detection labels or fluorescent groups are different, and 2 types of probes with different base sequences), then there are at least 1 signal acquisition includes 2 signal channel acquisitions. Among them, two signal channel acquisitions, that is, one signal acquisition performed under two signal channels (fluorescent channels) respectively. Those skilled in the art can predict the melting temperature of the reaction product according to the design of the primer-probe composition, and further predict the temperature required for the reaction product representing the target nucleic acid to generate a detectable signal, as well as the fluorescent channel in which it is located. Therefore, those skilled in the art can select an appropriate signal acquisition temperature and perform signal acquisition in an appropriate signal channel (fluorescent channel) according to the design of the primer-probe composition. In this way, in this embodiment, the two-dimensional analysis of fluorescence channel and melting temperature can be performed simultaneously in a single-tube reaction, that is, different targets can be detected by using the same fluorescence channel and different signal acquisition temperatures; or using different In the fluorescent channel, the target type detection can be realized by the product of the number of fluorescent channels and the signal acquisition temperature characteristics.

In some embodiments, the amplification reagents may also include reagents that promote PCR reactions, such as KCl, MgCl2, Tris-HCl, dithiothreitol (DTT), (NH4)2SO4, and the like. In some embodiments, besides the polymerase with polymerization activity, the amplification reagent also includes some other enzymes that act on nucleic acid, for example, exonuclease, endonuclease and the like.

In some embodiments, under the same signal channel, two adjacent signal channel acquisitions mean that the signal acquisition temperatures used for the two signal channel acquisitions are similar. For example, in some embodiments, under the same signal channel, the reaction product is placed at the first temperature for the first signal channel acquisition, then heated up to the second temperature for the second signal channel acquisition, and then continue to heat up When the third signal channel acquisition is carried out at the third temperature, the first signal channel acquisition and the second signal channel acquisition are two adjacent signal channel acquisitions, the second signal channel acquisition and the third signal channel acquisition It is collected for two adjacent signal channels, but the first signal channel acquisition and the third signal channel acquisition are not collected for two adjacent signal channels.

In some embodiments, the difference between the signal acquisition temperatures used in two adjacent signal channel acquisitions is more than 4°C, so as to achieve a difference of one target nucleic acid species contained in the signals obtained by two adjacent signal channel acquisitions under the same signal channel. , to avoid the situation that there is no difference in the target nucleic acid signals obtained by two adjacent signal channel acquisitions, thereby reducing the number of signal channel acquisitions and simplifying the operation steps.

In some embodiments, the signal collection temperature is 0-95°C. The selection of signal collection temperature can be determined by those skilled in the art according to the actual conditions of the designed primer-probe composition. For example, through the design of the primer-probe composition, those skilled in the art can expect that the melting temperature of the reaction product representing the target nucleic acid is T. If the signal of the target nucleic acid is to be detected, the signal acquisition temperature is ≤ T; If the signal of the target nucleic acid is detected, the signal collection temperature is >T.

In some embodiments, each signal collection includes m signal channel collections; said m is the number of detection labels of probes in the primer-probe composition. In order to facilitate the program design of the detection instrument, in some embodiments, if there are 2 kinds of probes in the primer-probe composition, each signal acquisition is performed under 2 signal channels for 2 signal channel acquisitions. In some embodiments, there are 5 kinds of targets in the sample to be tested, and the primer probe composition for the 5 kinds of targets contains 2 kinds of probes, the first probe detects 3 kinds of targets, and the second probe detects 3 kinds of targets. If there are 2 types of targets to be detected, only 3 signal acquisitions are required for the reaction product, and each signal acquisition is 2 signal channel acquisitions under 2 signal channels. In this way, under a certain signal channel, three signal acquisitions are carried out, but there are only two corresponding targets, and there is a difference in the number of target nucleic acid species contained in the signals obtained by two signal acquisitions with adjacent signal acquisition temperatures of 0. situation. In these embodiments, the number of signal acquisitions is set based on the detection channel with the largest number of detection targets, so that the number of signal acquisitions is less than the number of target nucleic acid types, reducing the number of signal acquisitions. Although the least number of signal acquisitions of other signal channels is not fully satisfied, the setting of the instrument program is more convenient, and those skilled in the art can choose according to actual needs. In some embodiments, according to the design of the primer-probe composition, those skilled in the art choose not to collect signals in channels without expected targets, thereby reducing the number of signal collections and simplifying the operation steps.

In certain embodiments, the above-mentioned method for detecting multiple target nucleic acids is a digital PCR detection method for multiple target nucleic acids. Before placing the reaction system under conditions that allow the nucleic acid polymerase to perform hybridization and extension reactions, it also includes: distributing the reaction system to more than 500 reaction units, each reaction unit containing the target nucleic acid of a sample to be tested Or do not contain the target nucleic acid of the sample to be tested. In some embodiments, the signal collection is collecting fluorescence signals by a camera. In some embodiments, the acquisition of the signal channel is to collect the fluorescence signal through a camera under the fluorescence signal channel.

In some embodiments, the primer probe composition, the sample to be tested (containing 2 targets) and the amplification reagent are mixed to obtain a reaction system, and then the reaction system is distributed to more than 500 reaction units to form countless small Liquid droplets, each small droplet contains at most one target nucleic acid of the sample to be tested, but contains components suitable for nucleic acid amplification, such as amplification reagents such as primer-probe composition and nucleic acid polymerase. All the droplets are then placed under conditions that allow nucleic acid polymerases to perform hybridization and extension reactions (in some embodiments, that is, PCR amplification conditions), and the resulting reaction products (amplification products) are subjected to a second temperature at a first temperature. After taking a photo for the first time, some small droplets glowed, and the positive signals of the two targets were obtained, and then the temperature was raised to the second temperature for the second photoshoot, and it was found that some of the small droplets in the first photoshoot did not emit light, Some small droplets still glow. Then, the small liquid droplets that still emit light in the second photographing are the first targets, and the small liquid droplets that do not emit light in the second photographing but glow in the first photographing are the second targets. According to the temperature and fluorescence channels used in the two photographs, the specific types of the two targets can be analyzed, and by analyzing the number of luminescent droplets in the second photograph, the number of the first target can be obtained, and the first photograph can be analyzed The difference between the number of luminous droplets and the number of luminescent droplets in the second photoshoot can be used to obtain the number of second targets.

In some embodiments, the above-mentioned conditions for allowing nucleic acid polymerase to perform hybridization and extension reactions include: pre-denaturation at about 85°C-about 105°C for 0-about 15 minutes; denaturation at about 85°C-about 105°C for about 1-about 60 seconds , about 40°C-about 75°C annealing and extension for about 3-about 90 seconds, 20-60 cycles; preferably, when the target in the sample to be tested is RNA, the amplification reagent also includes reverse transcriptase, for the The reaction conditions for the first PCR amplification of the reaction system include: about 30-about 65°C reverse transcription for about 2-about 30 minutes; about 85°C-about 105°C pre-denaturation for 0-about 15 minutes; about 85°C-about Denaturation at 105°C for about 1 to about 60 seconds, annealing at about 40°C to about 75°C and extension for about 3 to about 90 seconds, 20-60 cycles.

In some embodiments, the above-mentioned conditions allowing nucleic acid polymerases to perform hybridization and extension reactions include: 85°C-105°C pre-denaturation for 0-15 minutes; 85°C-105°C denaturation for 2-60 seconds, 40°C-75°C annealing and Extend for 10-90 seconds, 20-60 cycles. In some embodiments, when the target nucleic acid in the sample to be tested contains RNA, the amplification reagent further includes reverse transcriptase, and the conditions allowing the nucleic acid polymerase to perform hybridization and extension reactions include: 30-65°C reverse transcription 2 -30 minutes; pre-denaturation at 85°C-105°C for 0-15 minutes; denaturation at 85°C-105°C for 2-60 seconds, annealing and extension at 40°C-75°C for 10-90 seconds, 20-60 cycles.

In some embodiments, in the method for detecting multiple target nucleic acids, the above-mentioned primer probe composition includes a first probe and a first primer mixture; the first primer mixture includes at least two primer sets, different primers The groups specifically bind to different kinds of target nucleic acids, respectively. After the primer set in the first primer mixture specifically binds to its corresponding target nucleic acid, a pre-product is generated, and the pre-product contains a single-stranded pre-product that specifically binds to the first probe, and the single-stranded pre-product is combined with the The first probe specifically binds and extends ≥0 bases to form a double-stranded product, and the formation of the double-stranded product causes a detectable signal change.

In some embodiments, the single-stranded pre-products produced by different primer sets in the first primer mixture and their corresponding target nucleic acids are different, and the different single-stranded pre-products are different from the double-stranded products formed by the first probe. The double-stranded products have different melting temperatures. In some embodiments, the melting temperatures of different double-stranded products differ by more than 4°C.

The first probe refers to a type 1 probe with the same modified detection label, and its base sequence may be the same or different. That is, the number of types of probes is classified according to the number of types of markers they detect. In some embodiments, the signal generated by one probe can be collected in one detection channel (or fluorescent channel); the signal generated by two probes needs to be collected in two different detection channels. In some embodiments, the same detection labels do not mean that the modified detection groups are exactly the same. For example, the detection groups include fluorescent groups and quenching groups. As long as the fluorescent groups are the same, probes can also be The generated signal can be collected in one detection channel (or fluorescence channel).

"At least 2 primer sets" means that there are at least 2 types of primer sets, and each primer set targets different types of target nucleic acids, that is, each primer set can specifically bind to its corresponding target nucleic acid. In some embodiments, one primer set can specifically bind to one type of target nucleic acid regardless of type, then this type of target nucleic acid without type can also be considered as one type of target nucleic acid.

In certain embodiments, the primer set is a dual primer situation, ie, includes a pair of upstream primer and downstream primer for the same target nucleic acid. In some embodiments, the primer set is a single primer, that is, it contains only one kind of primer for a certain target nucleic acid, and the primer can specifically bind to its corresponding target nucleic acid but cannot specifically bind to other types of target nucleic acid.

In some embodiments, different types of primer sets may share primers, for example, two primer sets include the same primer. The same primer refers to the same sequence of the primer.

In this embodiment, the primer set for a certain target nucleic acid does not extend after the first single-stranded pre-product produced by it specifically binds to the probe (ie, extends by 0 bases). Those skilled in the art can realize the above-mentioned effect of "specific binding without extension" by designing the primer sequences and/or probe sequences in the primer set according to actual needs, for example, designing the first single Strand preproducts are bound at the 5' end of the probe, or, alternatively, the 3' end of the first single-stranded preproduct is designed to contain a region that does not pair complementary to the probe.

The melting temperature of oligonucleotides is related to factors such as the length and composition of oligonucleotides. Those skilled in the art can adjust the melting temperature of the oligonucleotide according to actual needs, for example, increasing the GC content of the oligonucleotide and making the length of the oligonucleotide longer can obtain a higher melting temperature. Therefore, those skilled in the art can adjust the sequence composition of primers and/or probes according to the actual situation, so that the single-stranded pre-products formed by different primers are different, and the positions where different single-stranded pre-products specifically bind to the first probe Different, so that the melting temperature of the different double-stranded products formed is different. That is to say, those skilled in the art can make different double-stranded products representing different targets according to the actual situation, and different double-stranded products have different melting temperatures, so that signals of different target nucleic acids can be obtained at different signal collection temperatures. In some embodiments, the design of the primers and probes can make the melting temperatures of different double-stranded products representing different targets differ by more than 4°C.

In some embodiments, the primer probe composition also includes a second probe and a second primer mixture; the base sequence of the second probe is different from that of the first probe, and the modified detection label are also different; the second primer mixture includes at least one primer set, and different primer sets specifically bind to different target nucleic acids.

The number of types of probes is classified according to the number of types of markers they detect. Different types of probes require different detection channels for signal acquisition due to their different detection labels. In some embodiments, different detection labels do not mean that the modified detection groups are completely different. For example, the detection groups include fluorescent groups and quenching groups, as long as the fluorescent groups are different (quenching groups can be The same or different), it is also possible to realize the signal acquisition of the signals generated by the two probes in the two detection channels (or fluorescent channels). In some embodiments, the increase in the number of types of probes can realize the signal detection of different target nucleic acids in different channels, and can realize the detection of more multiple target nucleic acids.

In this example, the appearance of "first probe" and "second probe" is not used to limit the role, nor is it used to limit that there are only two kinds of probes in this example. It is only used to distinguish "first probe" The "probe" and the "second probe" belong to different types of probes, and the detection labels modified by the two probes are different, and the base sequences are also different. In some embodiments, in order to detect more types of target nucleic acids, the primer probe composition for target nucleic acid detection also includes a third probe and a third primer mixture, a fourth probe and a fourth primer mixture, a third Five probe and fifth primer mixes, sixth probe and sixth primer mixes, or more probe and primer mixes. The detection labels modified by different probes are different, and the base sequences are also different; the primer sets in different primer mixtures are also different. That is to say, in order to realize more multiple target nucleic acid detection, those skilled in the art can design more primer sets corresponding to the target nucleic acid and more probes corresponding to the primer sets as required.

In some embodiments, the primer set in the second primer mixture specifically binds to its corresponding target nucleic acid to generate a pre-product, and the pre-product contains a single-stranded pre-product that specifically binds to the second probe, so The single-stranded pre-product specifically combines with the second probe and extends ≥ 0 bases to form a double-stranded product, and the formation of the double-stranded product causes a detectable signal change.

In some embodiments, different primer sets in the second primer mixture produce different single-stranded pre-products from their corresponding target nucleic acids, different single-stranded pre-products are different from double-stranded products formed by the second probe, and different double-stranded pre-products are different from the double-stranded products formed by the second probe. The chain products have different melting temperatures. In some embodiments, the melting temperatures of different double-stranded products differ by more than 4°C.

In some embodiments, in the primer probe composition, the first probe or the second probe is a sequence that does not specifically bind to any target nucleic acid, which includes a probe signal detection region (H), The sequences of the probe signal detection regions (H) of different probes are different from each other; the primer set includes a first primer and a second primer, and the first primer comprises a target sequence binding region 1; the second primer comprises a primer signal The detection region (h) and the target sequence binding region 2, and the primer signal detection region (h) is located at the 5' end of the target sequence binding region 2; the primer signal detection region (h) is a segment that does not specifically bind to any target nucleic acid , and the probe signal detection region (H) of the corresponding probe has part or all of the same sequence; the sequences of the primer signal detection region (h) of the second primer in different primer sets are different from each other.

In some embodiments, the target sequence binding region 1 and the target sequence binding region 2 specifically bind to different positions of the target nucleic acid respectively.

In some embodiments, the primer signal detection region (h) has part or all of the same sequence as the probe signal detection region (H) of the probe, which means that the reverse complementary sequence (h) of the primer signal detection region (h) '), capable of specifically binding to part or all of the sequence of the probe signal detection region (H) of the probe.

In some embodiments, the above-mentioned first primer and second primer specifically combine with the target nucleic acid to generate a pre-product, and the pre-product contains a reverse complementary sequence (h') having a primer signal detection region (h) The single-stranded pre-product, the reverse complementary sequence (h') of the single-stranded pre-product is specifically combined with the probe signal detection region (H) of the probe and extended ≥ 0 bases to form a double-stranded product, so The formation of the double-stranded product causes a detectable signal change.

In some embodiments, the sequences of the primer signal detection regions (h) of the second primers in different primer sets are different from each other, that is, the bases of the sequences of the primer signal detection regions (h) of the second primers in different primer sets Different and/or different in length, so that the sequence of the reverse complementary sequence (h') of different single-stranded preproducts is different, and different single-stranded preproducts specifically bind to the probe and extend 0, or > 0 bases to form Different double-stranded products, the melting temperature of different double-stranded products can be separated from each other; or, different single-stranded pre-products are respectively combined with different types of probes and extended ≥ 0 bases to form different double-stranded products , the melting temperatures of different double-stranded products cannot be separated from each other, but due to the different types of probes of different double-stranded products, they can be distinguished through different detection channels. That is, different types of target nucleic acids can be distinguished by melting temperature and/or detection channels to achieve multiple detection.

In some embodiments, there is an interval of 0-20 bases between the primer signal detection region (h) of the second primer and the target sequence binding region 2 .

In some embodiments of the present invention, in the above-mentioned primer probe composition for target nucleic acid detection, the first probe or the second probe further comprises a primer anchoring region (A'); the first primer mixture Or the first primer of at least one primer set in the second primer mixture also includes a probe anchor region (A), and the probe anchor region (A) is located at the 5' end of the target sequence binding region 1; The probe anchor region (A) does not specifically bind to any target nucleic acid but specifically binds to the primer anchor region (A').

The first primer and the second primer in the above primer set specifically combine with the target nucleic acid to produce a pre-product, which contains a reverse complementary primer with a probe anchoring region (A) and a primer signal detection region (h). The single-stranded pre-product of the sequence (h'), the probe anchor region (A) and the reverse complementary sequence (h') of the single-stranded pre-product, respectively, with the primer anchor region (A') of the probe, the probe After the needle signal detection region (H) specifically binds and extends ≥ 0 bases, a double-stranded product is formed, and the formation of the double-stranded product causes the probe to produce a detectable signal change.

By adjusting the probe anchor region (A) of the first primer in different primer sets, and/or the sequence composition and/or length of the primer signal detection region (h) of the second primer in different primer sets, different primer sets Different from the single-stranded pre-product produced by its target nucleic acid, different single-stranded pre-products have different annealing temperatures generated by specific binding to the probe, and the melting temperature of the double-stranded product formed after specific binding and extension ≥ 0 bases The lower the annealing temperature required to achieve the specific binding of a single-stranded pre-product generated by a certain primer set and its target nucleic acid to the probe, the single-stranded pre-product specifically binds to the probe and extends ≥ 0 bases The melting temperature of the double-stranded product formed by the group is higher.

In some embodiments, the sequences of the target sequence binding regions 1 of the first primers in different primer sets are different; the sequences of the probe anchor regions (A) of the first primers in different primer sets can be the same or different; preferably , there is an interval of 0-20 bases between the probe anchor region (A) of the first primer and the target sequence binding region 1;

In some embodiments of the present invention, in the above-mentioned primer-probe composition for target nucleic acid detection, in the first primer mixture or the second primer mixture, the second primer in at most one primer set also includes an extension resistance A blockage region (M), the extension blockage region (M) is located at the 5' end of the primer signal detection region (h), and the extension blockage region (M) and its complementary sequence are not combined with any probe or any Target nucleic acid specific binding.

The above-mentioned first primer and the second primer containing the extension blocking region (M) respectively specifically bind to the target nucleic acid to generate a pre-product, and the pre-product contains a reverse complementary sequence with a primer signal detection region (h) ( h'), the reverse complementary sequence (h') of the single-stranded pre-product specifically binds to the probe and extends 0 bases to form a double-stranded product (due to the existence of the extension block, The single-stranded pre-product specifically binds to the probe without elongation), thereby causing the probe to produce a detectable signal change.

"First primer mix", "second primer mix", etc. are used for descriptive purposes only to distinguish defined objects. Both represent a set of primer mixtures that specifically bind to the probe, including a variety of primer sets, the second primer in these primer sets contains the reverse complementary sequence (h) of the signal detection region (h) of the primer '), all of which can specifically bind to the same probe. However, the types of probes to which the primer sets specifically bind differ between the "first primer mix" and the "second primer mix".

In some embodiments, in a set of primer mixtures that specifically bind to the same probe, at most the second primer in one primer set contains an extension block region (M), and this primer set is specific to the target nucleic acid. The single-stranded pre-product produced after binding has the highest annealing temperature required for specific binding to the probe, and the lowest melting temperature of the double-stranded product produced after specific binding to the probe without extension.

In the present invention, "first primer" and "second primer" are only used for descriptive purposes to distinguish defined objects, and do not limit the order or priority in any way. For example, in the above-mentioned primer-probe composition, the structures of the first primer and the second primer of the primer set can be interchanged, for example, the first primer comprises a primer signal detection region (h) and a target sequence binding region, and the second primer comprises a target Sequence binding region; as another example, the first primer comprises a primer signal detection region (h) and a target sequence binding region, and the second primer comprises a probe anchor region (A) and a target sequence binding region. In some embodiments, the "first primer" is also called "forward primer", and the "second primer" is also called "reverse primer".

In the present invention, the probe is a freely designed sequence that does not pair with any target nucleic acid, and the probe is modified with a detection label. Preferably, the detection label includes a first detection group and a second detection group, and the first detection group and the second detection group produce a signal change through a change in distance; preferably, the first detection group The interval between the detection group and the second detection group is 3-250 angstroms; preferably, the interval is 3-201 angstroms; more preferably, the interval is 3-140 angstroms; and/or, the first detection group is A fluorescent reporter group, the second detection group is a quenching group or other modification groups capable of producing signal changes with the first detection group through fluorescence resonance energy transfer.

In the present invention, the positions of the first detection group and the second detection group on the probe make the reverse complementary sequence (h') of the single-stranded pre-product specifically bind to the probe (P) and extend ≥ After 0 bases, a double-stranded product is formed, as long as the formation of the double-stranded product can lead to a change in the position of the first detection group and the second detection group, it can cause the probe to produce a detectable signal change. The positions of the first detection group and the second detection group may be interchanged.

In some embodiments, when there is no target nucleic acid to be detected, no single-stranded pre-product specifically binds to the probe, and the probe is in a single-stranded state or other secondary structures. At this time, the first detection group and the second detection group The distance between the groups is relatively close, and the efficiency of fluorescence resonance energy transfer is high; if the first primer contains a probe anchor region (A), when there is no target nucleic acid to be detected, even if the primer anchor region of the probe (A') is complementary to the probe anchor region (A) in the first primer, but other parts of the probe are in a single-stranded state or other secondary structures. At this time, the first detection group and the second detection group The distance between them is still relatively short, and the efficiency of fluorescence resonance energy transfer is relatively high. When the target nucleic acid to be detected exists, the first primer and the second primer specifically bind to the target sequence and extend to generate a double-stranded amplification product, wherein one single-stranded amplification product specifically binds to the probe to form a double-stranded product, At this time, the distance between the first detection group and the second detection group becomes longer, and the efficiency of fluorescence resonance energy transfer decreases, so that the fluorescence signal changes and can be detected by the instrument.

In some embodiments, the primer probe composition, the sample to be tested, and the amplification reagent are mixed to obtain a reaction system, and then the reaction system is placed under conditions that allow nucleic acid polymerase to perform hybridization and extension reactions to obtain reaction products, Specifically include the following:

Under conditions that allow nucleic acid polymerase to perform hybridization and extension reactions, perform PCR amplification to obtain pre-products: the forward primer (F1) and reverse primer (R1) specifically bind to target sequence 1 and extend to generate a double-stranded pre-product. Amplified product, the 5' end to the 3' end of a single-stranded pre-amplified product (S1) is the probe anchor region (A1), target sequence, and the reverse complementary sequence of the primer signal detection region (h1') , the reverse complementary sequence (M') of the extension block region; the forward primer (F2) and the reverse primer (R2) respectively specifically bind to the target sequence 2 and extend to generate double-stranded pre-amplified products, one of which is single-stranded The 5' end to the 3' end of the pre-amplification product (S2) is followed by the probe anchor region (A2), the target sequence, and the reverse complementary sequence (h2') of the primer signal detection region; optional, if there is a positive The forward primer (F3) and reverse primer (R3), the forward primer (F3) and the reverse primer (R3) specifically bind to the target sequence 3 and extend to generate double-stranded pre-amplified products, one of which is single-stranded pre-amplified The 5' end to the 3' end of the amplification product (S3) is the probe anchor region (A3), the target sequence, and the reverse complementary sequence (h3') of the primer signal detection region;

Put the above-mentioned pre-product under conditions that allow nucleic acid polymerase to carry out hybridization and extension reactions (constant temperature incubation, or PCR amplification conditions of several high and low temperature cycles), and obtain the reaction product: at this time, the probe signal of the probe (P) The detection region (H) is reverse complementary to the reverse complementary sequence (h1') of the primer signal detection region of the single-stranded preamplification product (S1), and the primer anchor region (A1') of the probe (P) is paired with the single The probe anchor region (A1) at the 5' end of the strand preamplification product (S1) is reverse-complementary paired to obtain a hybrid double-stranded product (D1) formed with the probe (P), whose melting temperature is T1, while the single-stranded The reverse complementary sequence (M') of the extension block region at the 3' end of the preamplified product (S1) is not complementary to any part of the probe (P) or target sequence; at this time, the probe signal of the probe (P) is detected The region (H) is complementary to the reverse complementary sequence (h2') of the primer signal detection region at the 3' end of the single-stranded preamplification product (S2), and the primer anchor region (A2') of the probe is compatible with the single-stranded preamplified The probe anchor region (A2) at the 5' end of the product (S2) is reverse-complementary paired, and the 3' end of the single-stranded preamplified product (S2) can continue to extend to the 5' end of the probe (P) to obtain a probe Part or all of the reverse complementary sequence of (P), that is, to complete the secondary amplification using the single-stranded pre-amplified product (S2) as a primer and the probe (P) as a template, and obtain the formation of the probe (P) Amplify the double-stranded product (D2), its melting temperature is T2, T2>T1; Optionally, if there is a forward primer (F3) and a reverse primer (R3), the probe signal of the probe (P) The detection region (H) is complementary to the reverse complementary sequence (h3') of the primer signal detection region at the 3' end of the single-stranded preamplified product (S3), and the primer anchor region (A3') of the probe is matched with the single-stranded preamplified product (S3). The probe anchor region (A3) at the 5' end of the amplification product (S3) is reverse-complementary paired, and the 3' end of the single-stranded pre-amplification product (S3) can continue to extend to the 5' end of the probe (P) to obtain a probe Part or all of the reverse complementary sequence of the needle (P), that is, to complete the secondary amplification with the single-stranded pre-amplification product (S3) as the primer and the probe (P) as the template, and obtain the formation of the probe (P) The amplified double-stranded product (D3) has a melting temperature of T3, and T3>T2.

In some embodiments, the reaction product is placed at n different signal acquisition temperatures for n times of signal acquisition, and then analyzed under the same signal channel, whether the signals obtained by two signal acquisitions with adjacent signal acquisition temperatures exist or not The difference, determine the presence or absence of the target nucleic acid in the sample to be tested, specifically include the following:

The first signal acquisition is performed at the first signal acquisition temperature (t1, t1≤T1), since the single-stranded amplification products S1, S2 and S3 respectively form a full or partial double-stranded structure D1, D2 with the probe (P) , D3, compared with the state of the probe (P) before PCR, the distance between the first detection group and the second detection group becomes farther, and the efficiency of fluorescence resonance energy transfer is lower, so that the fluorescence signal changes, which can be detected by detected by the instrument; then the temperature is raised to the second signal acquisition temperature (t2, T1<t2≤T2) for the second signal acquisition, the amplification formed by the single-stranded pre-amplification product (S1) and the probe (P) The double-stranded product (D1) cannot form a double-stranded structure because the second signal collection temperature (t2) is higher than the melting temperature of the amplified double-stranded product (D1), and no fluorescent signal is generated, while the single-stranded pre-amplified product (S2 ) and the amplified double-stranded product (D2) formed by the probe (P) and the amplified double-stranded product (D3) formed by the single-stranded pre-amplified product (S3) and the probe (P) are still in a double-stranded state, which can be The fluorescent signal is detected by the instrument; optionally, if there is a forward primer (F3) and a reverse primer (R3), then the temperature is raised to the third signal acquisition temperature (t3, T2<t3≤T3) for the third In the second signal acquisition, the amplified double-stranded product (D2) formed by the single-stranded pre-amplification product (S2) and the probe (P) is higher than the melting of the amplified double-stranded product (D2) due to the third signal acquisition temperature (t3) temperature and cannot form a double-stranded structure, no fluorescent signal is generated, and the amplified double-stranded product (D3) formed by the single-stranded pre-amplified product (S3) and the probe (P) is still in a double-stranded state and can be detected by the instrument to the fluorescent signal.

In some embodiments, the reaction product is placed at n different signal acquisition temperatures for n times of signal acquisition, and then analyzed under the same signal channel, whether the signals obtained by two signal acquisitions with adjacent signal acquisition temperatures exist or not The difference, determine the presence or absence of the target nucleic acid in the sample to be tested, specifically include the following:

Optionally, if there is a forward primer (F3) and a reverse primer (R3), the first signal acquisition is performed at the first signal acquisition temperature (t3, T2<t3≤T3), and the single-stranded pre-amplification product ( S1) The amplified double-stranded product (D1) formed with the probe (P) and the amplified double-stranded product (D2) formed by the single-stranded pre-amplified product (S2) and the probe (P) are due to the first signal acquisition temperature (t3) is higher than the melting temperature of the amplified double-stranded products (D1 and D2) and cannot form a double-stranded structure, while the amplified double-stranded product (D3) formed by the single-stranded pre-amplified product (S3) and the probe (P) ) Compared with the state of the probe (P) before PCR, the distance between the first detection group and the second detection group becomes farther, and the efficiency of fluorescence resonance energy transfer is lower, so that the fluorescence signal changes and can be detected by the instrument Then the temperature is lowered to the second signal collection temperature (t2, T1<t2≤T2) for the second signal collection, the amplified double-strand formed by the single-stranded pre-amplification product (S1) and the probe (P) The product (D1) cannot form a double-stranded structure because the second signal collection temperature (t2) is higher than the melting temperature of the amplified double-stranded product (D1), and no fluorescent signal is generated, while the single-stranded pre-amplified product (S2) and For the amplified double-stranded product (D2) formed by the probe (P), compared with the state of the probe (P) before PCR, the distance between the first detection group and the second detection group becomes farther, and the fluorescence resonance energy The transfer efficiency is low, so that the fluorescent signal changes and can be detected by the instrument. The amplified double-stranded product (D3) formed by the single-stranded pre-amplification product (S3) and the probe (P) is still double-stranded and can be detected by the instrument. The instrument detects the fluorescent signal; then lower the temperature to the third signal acquisition temperature (t1, t1≤T1) for the third signal acquisition, the amplification formed by the single-stranded pre-amplification product (S1) and the probe (P) For the double-stranded product (D1), compared with the state of the probe (P) before PCR, the distance between the first detection group and the second detection group becomes longer, and the fluorescence resonance energy transfer efficiency is lower, so that the fluorescence signal occurs The change can be detected by the instrument, the amplified double-stranded product (D3) formed by the single-stranded pre-amplification product (S3) and the probe (P) and the double-stranded product (D3) formed by the single-stranded pre-amplified product (S2) and the probe (P) The amplified double-stranded product (D2) is still double-stranded and can be detected by the instrument as a fluorescent signal.

A second aspect of the present invention provides a device for detection of various target nucleic acids, comprising:

The reaction liquid containing part is used to accommodate several micro-liquids, each of which contains a reaction reagent, and some of the micro-liquids also contain one of the first analyte or the second analyte;

a temperature regulating part, for regulating the temperature of the micro liquid in the reaction liquid containing part;

a signal detection part, used to detect the signal generated by the micro-liquid in the reaction solution containing part;

A control unit, the control unit controls the temperature adjustment unit to adjust the temperature of the micro-liquids in the reaction liquid container, so that several micro-liquids containing the first analyte or the second analyte generate signals at the same time;

The control part controls the temperature regulating part to adjust the temperature of the micro-liquid to t1, and several micro-liquids containing the first analyte and several micro-liquids containing the second analyte generate signals, forming first mixed signal;

The control part controls the temperature regulating part to adjust the temperature of the micro-liquid to t2, and only a few micro-liquids containing the second analyte generate signals to form a second signal;

Wherein, t1<t2; the control part controls the signal detection part to collect the signal generated by the micro-liquid at the temperature t1 and t2, and output the first mixed signal and the second signal;

A signal analysis unit, the signal analysis unit calculates the first signal according to the first mixed signal and the second signal collected by the signal detection unit.

In some embodiments, the control part controls the temperature adjustment part to adjust the temperature of the micro-liquid in the reaction liquid containing part to t3, and several micro-liquids containing the first analyte, containing the first Several microfluids containing the second analyte and several microfluids containing the third analyte generate signals to form a second mixed signal, wherein t3<t1, and the difference between t3 and t1 is more than 4°C.

In some embodiments, the difference between t1 and t2 is more than 4°C.

In some embodiments, the difference between t1 and t2 is 4°C.

In some embodiments, the signal analysis unit subtracts the first mixed signal from the second signal to obtain the first signal.

In some embodiments, when collecting the first mixed signal and the second signal, several of the micro-liquids are spread on the bottom of the reaction solution container, and always maintain the same position state.

In some embodiments, the first signal is the fluorescence signal generated by the microfluid containing the first analyte, and the second signal is the fluorescence generated by the microfluid containing the second analyte signal, the first mixed signal is a fluorescent signal generated by the microfluid containing the first analyte and the microfluid containing the second analyte.

In some embodiments, the signal analysis part subtracts the fluorescent signal generated by the microliquid containing the second analyte at the same position from the first mixed signal generated by the microliquid to obtain the the first signal.

In some embodiments, the signal analysis unit subtracts the second mixed signal generated by the micro-liquid from the micro-liquid containing the first analyte and the micro-liquid containing the second analyte at the same position. The first mixed signal generated by the micro-fluid is used to obtain a third signal, and the third signal is a fluorescence signal generated by the micro-fluid containing the third analyte. In some embodiments, each of the micro-liquids contains a reaction reagent, the reaction reagent includes an amplification substance for amplifying nucleic acid and a labeling substance for labeling nucleic acid, and the amplification substance can amplify different Nucleic acid molecules, labeled substances can combine with nucleic acids at a certain temperature to produce detectable signals.

In some embodiments, the reaction reagent includes a primer probe composition and an amplification reagent; the amplification reagent includes nucleic acid polymerase and dNTPs; the primer probe composition includes a first probe and a first primer mixture ;

The first primer mixture includes at least two primer sets, and different primer sets specifically bind to different target nucleic acids;

After the primer set in the first primer mixture specifically binds to its corresponding target nucleic acid, a pre-product is generated, and the pre-product contains a single-stranded pre-product that specifically binds to the first probe, and the single-stranded pre-product is combined with the The first probe specifically binds and extends ≥ 0 bases to form a double-stranded product, and the formation of the double-stranded product causes a detectable signal change;

In some embodiments, the single-stranded pre-products produced by different primer sets in the first primer mixture and their corresponding target nucleic acids are different, and the different single-stranded pre-products are different from the double-stranded products formed by the first probe. The melting temperatures of the double-stranded products are different; in some embodiments, the melting temperatures of different double-stranded products differ by more than 4°C.

In some embodiments, the primer probe composition also includes a second probe and a second primer mixture;

The base sequence of the second probe is different from that of the first probe, and the modified detection label is also different; the second primer mixture includes at least one primer set, and different primer sets are respectively matched with different target Nucleic acid specific binding;

In some embodiments, the primer set in the second primer mixture specifically binds to its corresponding target nucleic acid to generate a pre-product, and the pre-product contains a single-stranded pre-product that specifically binds to the second probe, so The single-stranded pre-product specifically binds to the second probe and extends ≥ 0 bases to form a double-stranded product, and the formation of the double-stranded product causes a detectable signal change;

In some embodiments, different primer sets in the second primer mixture produce different single-stranded pre-products from their corresponding target nucleic acids, different single-stranded pre-products are different from double-stranded products formed by the second probe, and different double-stranded pre-products are different from the double-stranded products formed by the second probe. The melting temperatures of the chain products are different; preferably, the melting temperatures of different double-chain products differ by more than 4°C.

In some embodiments, the first probe or the second probe is a sequence that does not specifically bind to any target nucleic acid, which includes a probe signal detection region (H), and a probe signal detection region of different probes The sequences of (H) are different from each other;

The primer set includes a first primer and a second primer, the first primer includes a target sequence binding region 1; the second primer includes a primer signal detection region (h) and a target sequence binding region 2, and the primer signal detection region (h) is located at the 5' end of the target sequence binding region 2; the primer signal detection region (h) is a section that does not specifically bind to any target nucleic acid and has a part with the probe signal detection region (H) of the corresponding probe or all identical sequences; the sequences of the primer signal detection regions (h) of the second primers in different primer sets are different from each other;

Preferably, the first probe or the second probe further comprises a primer anchor region (A'); the first primer of at least one primer set in the first primer mixture or the second primer mixture further comprises a probe Needle anchoring region (A), described probe anchoring region (A) is positioned at the 5' end of target sequence binding region 1; Described probe anchoring region (A) is not specifically combined with any target nucleic acid, but with The primer anchor region (A') specifically binds;

Preferably, in the first primer mixture or the second primer mixture, the second primer in at most one primer set further includes an extension block region (M), and the extension block region (M) is located in the primer signal detection region. The 5' end of region (h), said extension block region (M) and its complement, neither specifically binds to any probe or to any target nucleic acid.

Beneficial effect

Compared with the prior art, the primer probe composition and digital PCR detection method of the present invention have the following advantages:

(1) The method is simple: the method of the present invention can realize at least 8 multiple reactions on the digital PCR, and different targets can be distinguished by taking photos for a limited number of times to distinguish the presence or absence of fluorescence, and the requirements for the fluorescence recognition of digital PCR are low , easy to operate and save time;

(2) Multiple detection: the method of the present invention can simultaneously analyze the two dimensions of fluorescence channel and melting temperature in a single-tube reaction, that is, using the same fluorescence channel, different signals can be collected at different temperatures to detect different targets; or Using different fluorescent channels, the target type detection can be realized by the product of the number of fluorescent channels and the signal acquisition temperature characteristics;

(3) Low fluorescent background: each fluorescent channel uses only one probe, which can be distinguished by the different melting temperatures of the amplified products, which greatly reduces the fluorescent background in the PCR reaction and improves the reaction sensitivity;

(4) The melting temperature can be adjusted: the method of the present invention utilizes the different melting temperatures of the secondary amplification products to distinguish, so by adjusting the length or sequence of the primer signal detection region (h), or adjusting the primer signal detection region (h) ) the reverse complementary position to the full signal detection region (H) of the probe (P), thereby increasing or decreasing the melting temperature of the secondary amplified double-stranded product formed with the probe (P);

(5) Good inclusiveness: the primer probe design method of the present invention has two parts of reverse complementary pairing with the target sequence, i.e. forward primer (F) and reverse primer (R), compared with Taqman hydrolysis probe method needs The three parts are reverse-complementary paired with the template. When there are many highly variable regions in the target sequence, such as viral or bacterial genomes, the primer probe design method of the present invention is more tolerant and less difficult to design;

(6) Low cost: the number of fluorescent probes in the reagent is reduced, which greatly reduces the cost of the reagent, making large-scale promotion possible;

(7) Single-tube reaction: less consumption of samples, especially suitable for the detection of rare samples, can greatly increase the concentration of samples added to the detection reaction, and improve detection sensitivity. the possibility

(8) Easy to operate: only need to use a digital PCR instrument for detection, without subsequent steps such as capillary electrophoresis or nucleic acid hybridization, etc., and at the same time, the preparation is simple, and no operation is required after the machine is installed;

(9) It is not easy to cause pollution: the method of the present invention is completely closed after adding the sample, and there is no need to open the cover for subsequent detection, which greatly reduces the possibility of causing pollution to the experimental environment;

(10) High sensitivity: the method of the present invention has very low requirements on the length of the target sequence. When the target type is a short fragment of nucleic acid, such as free nucleic acid, a shorter target sequence length has higher sensitivity in detection;

(11) Wide range of application: the method of the present invention can be applied to nucleic acid detection of various sample types, including serum samples, plasma samples, whole blood samples, sputum samples, swab samples, lavage fluid samples, fresh tissue samples , formalin-fixed paraffin-embedded tissue (FFPE), etc.

Description of drawings

Fig. 1A is a signal diagram collected at 50°C in Example 1; Fig. 1B is a signal diagram collected at 68°C in Example 1;

Figure 2A is a signal diagram collected at 50°C in Example 2; Figure 2B is a signal diagram collected at 68°C in Example 2; Figure 2C is a signal diagram collected at 77°C in Example 2;

Figure 3A is a signal diagram collected at 70°C in Example 3; Figure 3B is a signal diagram collected at 50°C in Example 3;

Figure 4A is a signal diagram collected at 77°C in Example 4; Figure 4B is a signal diagram collected at 70°C in Example 4; Figure 4C is a signal diagram collected at 50°C in Example 4;

Fig. 5A is a schematic diagram of a variety of target nucleic acid detection devices disclosed in one embodiment of the present application; Fig. 5B is a schematic diagram of a variety of target nucleic acid detection devices disclosed in another embodiment of the present application; Fig. 5C is a schematic diagram of a variety of target nucleic acid detection devices disclosed in another embodiment of the present application Structural diagram of an orifice plate composed of multiple reaction liquid storage parts.

Detailed ways

The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be presented more clearly. Those skilled in the art should understand that these specific implementations and examples are used to illustrate the present invention, not to limit the present invention.

In the following examples, the D600 fully automatic digital PCR analysis system and reagent consumables of Mike Biological Co., Ltd. are used for detection and data analysis. The digital PCR analysis system disperses the reaction system containing samples, primer probe compositions and amplification reagents. Perform amplification and detection in 4 reaction wells (each reaction well contains multiple small droplets, each small droplet is 1 reaction unit), in order to avoid redundancy, the accompanying drawings of the following examples are all provided Droplet results plot for 1 reaction well.

Example 1

(1) The dual detection primer probe system for detecting carbapenem-resistant gene VIM mutation and carbapenem-resistant gene KPC mutation is shown in Table 1:

The base sequence of table 1 embodiment 1 primer probe

Among the above primer probes:

The forward primer F1-1 (SEQ ID NO: 2), the reverse primer R1-1 (SEQ ID NO: 3), are all designed for the specificity of the gene VIM mutation target sequence resistant to carbapenem antibiotics Primer, the full length of F1-1 is 39bp, the 1st to 23rd bases at the 3' end are the target sequence binding region, and the 1st to 13th bases at the 5' end are compatible with the probe P1 (SEQ ID NO: 1) The 1st to 13th nucleotide sequence at the 5' end of the R1-1 primer is 49 bp in full length, the 1st to 25th nucleotide sequence at the 3' end is the target sequence binding region, and the 6th to 13th nucleotide sequence at the 5' end is the target sequence binding region. The 21st base is the same as the 16th to 31st bases at the 5' end of the probe P1 (SEQ ID NO: 1), and the 1st to 5th bases at the 5' end are amplification retardation district.

The forward primer F1-2 (SEQ ID NO: 4), the reverse primer R1-2 (SEQ ID NO: 5), are all designed for the specificity of the gene KPC mutation target sequence resistant to carbapenem antibiotics Primer, the full length of F2 is 37bp, the 1st to 21st bases of its 3' end are the target sequence binding region, and the 1st to 13th bases of its 5' end are compatible with the 5th base of probe P1 (SEQ ID NO: 1) The 1st to 13th base sequences at the 'end are reverse complementary; the R1-2 primer is 35 bp in length, the 1st to 22nd bases at the 3' end are the target sequence binding region, and the 1st to 10th bases at the 5' end The 32nd to 41st base sequences of the 5' end of the probe probe P1 (SEQ ID NO: 1) are identical.

(2) The detection of two kinds of target nucleic acids was carried out by using the above-mentioned primer-probe system, and the reaction system used in the detection is shown in Table 2.

The detection reaction system of table 2 embodiment 1

Reagent components		concentration
2×PCR Reaction Buffer	1×
DNA Polymerase	2U
Forward primer (F1-1)	500nM
Reverse primer (R1-1)	100nM
Forward primer (F1-2)	500nM

Reverse primer (R1-2)	100nM
Probe (P1)	400nM
Gene VIM mutation nucleic acid template resistant to carbapenem antibiotics	2000 copies
Carbapenem-resistant gene KPC mutation nucleic acid template	1000 copies
Ultra-pure water	Add to 20μL

Note: 2X PCR Reaction Buffer includes: 3mM MgCl ₂ , 30mM Tris-HCl at pH 8.3, 0.5mM dNTP and 70mM (NH ₄ ) ₂ SO ₄

(3) The specific operation steps during detection are as follows:

Sample preparation: simultaneously use the nucleic acid template of carbapenem-resistant gene VIM mutation and the nucleic acid template of carbapenem-resistant gene KPC mutation as positive samples for detection. Pure water was used as a no-template control (NTC).

Reaction preparation: after the sample preparation is completed, configure the reaction system according to the ratio described in Table 2;

Digital PCR amplification and signal collection: Seal the cap of the digital PCR tube and mix the sample gently, then centrifuge briefly and let it stand at room temperature for 5 minutes. The digital PCR tube was placed in the hand-held centrifuge again, and after a brief centrifugation, it was transferred to the sample rack of the digital PCR instrument (D600 fully automatic digital PCR analysis system of Mike Biological Co., Ltd.). The amplification and lighting programs used are: pre-denaturation at 95°C for 2 minutes; denaturation at 94°C for 10 seconds, annealing and extension at 56°C for 30 seconds, a total of 45 cycles; the first signal acquisition at 50°C; the second at 68°C Signal Acquisition.

Data analysis: Interpret the results through positive droplets at different signal collection temperatures.

Figure 1A is a droplet diagram at the first signal acquisition temperature (50°C), and it can be seen that there are positive droplets (bright spots are positive droplets) that are different from the background; Figure 1B is at the second signal acquisition temperature (68°C). ℃), it can be seen that there are positive droplets that are different from the background, and the number of positive droplets is reduced compared with that in Figure 1A (part of the positive droplets in Figure 1A, in the corresponding position in Figure 1B no positive droplets).

The positive droplets that still exist in Figure 1B are the signals of the gene KPC mutation-positive samples resistant to carbapenem antibiotics. By analyzing the number of positive droplets, the content of the gene KPC mutation-positive samples can also be determined; The positive droplets that do not exist in 1B but exist in Figure 1A are the signal of the VIM mutation sample resistant to carbapenem antibiotics. By analyzing the difference between the positive droplets in Figure 1A and Figure 1B, it can also be Determine the content of gene VIM mutation positive samples.

It can be seen from FIG. 1A and FIG. 1B that the detection method of the present invention can realize the detection of various target nucleic acids. The realization of multiple detection does not only depend on the fluorescent channel, but also uses different melting temperatures in the same fluorescent channel (that is, the same signal channel), and can distinguish between different targets by taking a limited number of photos to distinguish the presence or absence of fluorescence. The fluorescent recognition of digital PCR has low requirements, simple operation and time saving.

Example 2

(1) The triple primer probe system for detecting carbapenem-resistant gene VIM mutation and carbapenem-resistant gene KPC mutation and carbapenem-resistant gene OXA-48 mutation is as follows: Table 3 shows:

The base sequence of table 3 embodiment 2 primer probe

Among the above primer probes:

The forward primer F2-1 (SEQ ID NO: 2), the reverse primer R2-1 (SEQ ID NO: 3), are all designed for the specificity of the gene VIM mutation target sequence resistant to carbapenem antibiotics Primer, the full length of F2-1 is 39bp, the 1st to 23rd bases of its 3' end are the target sequence binding region, and the 1st to 13th bases of its 5' end are connected with the probe P2 (SEQ ID NO: 1) The 1st to 13th nucleotide sequence at the 5' end of the R2-1 primer is 49 bp in full length, and the 1st to 25th nucleotide sequence at its 3' end is the target sequence binding region, and the 6th to 13th nucleotide sequence at its 5' end is The 21st base has the same sequence as the 16th to 31st bases at the 5' end of the probe P2 (SEQ ID NO: 1), and the 1st to 5th bases at the 5' end are amplification retardation district.

The forward primer F2-2 (SEQ ID NO: 4), the reverse primer R2-2 (SEQ ID NO: 5), are all designed for the specificity of the gene KPC mutation target sequence resistant to carbapenem antibiotics Primer, the full length of F2-2 is 37bp, the 1st to 21st bases at the 3' end are the target sequence binding region, and the 1st to 13th bases at the 5' end are compatible with the probe P2 (SEQ ID NO: 1) The 1st to 13th nucleotide sequences at the 5' end of the primer are reverse complementary; the R2-2 primer is 35 bp in length, the 1st to 22nd nucleotides at its 3' end are the target sequence binding region, and the 1st to 13th bases at its 5' end The 10th base is completely identical to the 32nd to 41st base sequence of the 5' end of the probe probe P2 (SEQ ID NO: 1).

The forward primer F2-3 (SEQ ID NO: 6), the reverse primer R2-3 (SEQ ID NO: 7), are all designed for the gene OXA-48 mutation target sequence resistant to carbapenem antibiotics Specific primers, the full length of F2-3 is 35bp, the 1st to 19th bases at the 3' end are the target sequence binding region, and the 1st to 13th bases at the 5' end are compatible with the probe P2 (SEQ ID NO: 1) The 1st to 13th nucleotide sequence at the 5' end is reverse complementary; the R2-2 primer is 37 bp in full length, the 1st to 24th nucleotide sequence at its 3' end is the target sequence binding region, and the 5' end nucleotide sequence is The 1st to 10th bases are completely identical to the 1st to 10th bases of the 3' end of the probe probe P2 (SEQ ID NO: 1).

(2) The detection of three kinds of target nucleic acids was carried out by using the above-mentioned primer-probe system, and the reaction system used in the detection is shown in Table 4.

The detection reaction system of table 4 embodiment 2

Reagent components		concentration
2×PCR Reaction Buffer	1×
DNA Polymerase	2U
Forward primer (F2-1)	500nM
Reverse primer (R2-1)	100nM
Forward primer (F2-2)	500nM
Reverse primer (R2-2)	100nM
Forward primer (F2-3)	500nM
Reverse primer (R2-3)	100nM
Probe (P2)	400nM
Gene VIM mutation nucleic acid template resistant to carbapenem antibiotics	2400 copies
Carbapenem-resistant gene KPC mutation nucleic acid template	80 copies
Carbapenem-resistant gene OXA-48 mutation nucleic acid template	800 copies
Ultra-pure water	Add to 20μL

Note: 2X PCR Reaction Buffer includes: 3mM MgCl ₂ , 30mM Tris-HCl at pH 8.3, 0.5mM dNTP and 70mM (NH ₄ ) ₂ SO ₄

(3) The specific operation steps during detection are as follows:

Sample preparation: simultaneously use the nucleic acid template of carbapenem-resistant gene VIM mutation, the nucleic acid template of carbapenem-resistant gene KPC mutation and the nucleic acid template of carbapenem-resistant gene OXA-48 mutation The template is tested as a positive sample. Pure water was used as a no-template control (NTC).

Reaction preparation: after the sample preparation is completed, configure the reaction system according to the ratio described in Table 4;

Digital PCR amplification and signal collection: Seal the cap of the digital PCR tube and mix the sample gently, then centrifuge briefly and let it stand at room temperature for 5 minutes. The digital PCR tube was placed in the hand-held centrifuge again, and after a brief centrifugation, it was transferred to the sample rack of the digital PCR instrument (D600 fully automatic digital PCR analysis system of Mike Biological Co., Ltd.). The amplification and lighting programs used are: 95°C pre-denaturation for 2 minutes; 94°C denaturation for 10 seconds, 56°C annealing and extension for 30 seconds, a total of 45 cycles; 40°C constant temperature incubation for 3 minutes; 50°C for the first signal acquisition; The second signal acquisition was performed at 68°C; the third signal acquisition was performed at 77°C.

Data analysis: Interpret the results through positive droplets at different signal collection temperatures.

Figure 2A is a droplet diagram at the first signal acquisition temperature (50°C), and it can be seen that there are positive droplets that are different from the background (bright spots are positive droplets); Figure 2B is at the second signal acquisition temperature (68°C). ℃), it can be seen that there are positive droplets that are different from the background, and the number of positive droplets is reduced compared with that in Figure 2A (part of the positive droplets in Figure 2A, in the corresponding position in Figure 2B There are no positive droplets); Figure 2C is a droplet diagram at the third signal acquisition temperature (77°C), and it can be seen that there are positive droplets that are different from the background, and the number of positive droplets is reduced compared to Figure 2B (Some of the positive droplets in FIG. 2B have no positive droplets in the corresponding positions in FIG. 2C).

The positive droplets that still exist in Figure 2C are the signals of carbapenem-resistant gene OXA-48 mutation-positive samples. By analyzing the number of positive droplets, the number of gene OXA-48 mutation-positive samples can also be determined. content; the positive droplets that do not exist in Figure 2C but exist in Figure 2B are the signal of the gene KPC mutation resistant to carbapenem antibiotics, by analyzing the difference between the positive droplets in Figure 2B and Figure 2C , it is also possible to determine the content of gene KPC mutation-positive samples; the positive droplets that do not exist in Figure 2B but exist in Figure 2A are the signals of carbapenem-resistant gene VIM mutation samples. The difference between positive droplets in Figure 2A and Figure 2B can also determine the content of gene VIM mutation positive samples.

It can be seen from FIG. 2A-FIG. 2C that the detection method of the present invention can realize the detection of various target nucleic acids. The realization of multiple detection does not only depend on the fluorescent channel, but also uses different melting temperatures in the same fluorescent channel (that is, the same signal channel), and can distinguish between different targets by taking a limited number of photos to distinguish the presence or absence of fluorescence. The fluorescent recognition of digital PCR has low requirements, simple operation and time saving.

Example 3

(1) The dual detection primer probe system for detecting carbapenem-resistant gene VIM mutation and carbapenem-resistant gene OXA-48 mutation is shown in Table 5:

The base sequence of table 5 embodiment 3 primer probes

Among the above primer probes:

The forward primer F3-1 (SEQ ID NO: 9), the reverse primer R3-1 (SEQ ID NO: 10), are all designed for the specificity of the gene VIM mutation target sequence resistant to carbapenem antibiotics Primer, the full length of F3-1 is 39bp, the 1st to 23rd bases of its 3' end are the target sequence binding region, and the 1st to 13th bases of its 5' end are connected with the probe P3 (SEQ ID NO: 8) The 1st to 13th base sequence at the 5' end of the primer is reverse complementary; the R3-1 primer is 49 bp in length, the 1st to 25th base at its 3' end is the target sequence binding region, and the 6th to 13th base at its 5' end The 21st base has the same sequence as the 16th to 31st bases at the 5' end of probe P3 (SEQ ID NO: 8), and the 1st to 5th bases at the 5' end are amplification retardation district.

The forward primer F3-2 (SEQ ID NO: 11), the reverse primer R3-2 (SEQ ID NO: 12), are all designed for the gene OXA-48 mutation target sequence resistant to carbapenem antibiotics Specific primer, the full length of F3-2 is 35bp, the 1st to 19th bases at the 3' end are the target sequence binding region, and the 1st to 13th bases at the 5' end are compatible with the probe P3 (SEQ ID NO: 8) The 1st to 13th nucleotide sequence at the 5' end is reverse complementary; the R3-2 primer is 38 bp in full length, the 1st to 24th nucleotide sequence at its 3' end is the target sequence binding region, and the 5' end nucleotide sequence is The 1st to 11th bases are completely identical to the 32nd to 42nd bases at the 5' end of the probe probe P3 (SEQ ID NO: 8).

(2) The detection of two kinds of target nucleic acids was carried out by using the above-mentioned primer-probe system, and the reaction system used in the detection is shown in Table 6.

The detection reaction system of table 6 embodiment 3

Reagent components		concentration
2×PCR Reaction Buffer	1×
DNA Polymerase	2U
Forward primer (F3-1)	500nM
Reverse primer (R3-1)	100nM
Forward primer (F3-2)	500nM
Reverse primer (R3-2)	100nM
Probe (P3)	400nM
Gene VIM mutation nucleic acid template resistant to carbapenem antibiotics	10 copies
Carbapenem-resistant gene OXA-48 mutation nucleic acid template	100 copies
Ultra-pure water	Add to 20μL

Note: 2X PCR Reaction Buffer includes: 3mM MgCl ₂ , 30mM Tris-HCl at pH 8.3, 0.5mM dNTP and 70mM (NH ₄ ) ₂ SO ₄

(3) The specific operation steps during detection are as follows:

Sample preparation: simultaneously use the nucleic acid template of the carbapenem-resistant gene VIM mutation and the nucleic acid template of the carbapenem-resistant gene OXA-48 mutation as positive samples for detection. Pure water was used as a no-template control (NTC).

Reaction preparation: after the sample preparation is completed, configure the reaction system according to the ratio described in Table 6;

Digital PCR amplification and signal collection: Seal the cap of the digital PCR tube and mix the sample gently, then centrifuge briefly and let it stand at room temperature for 5 minutes. The digital PCR tube was placed in the hand-held centrifuge again, and after a brief centrifugation, it was transferred to the sample rack of the digital PCR instrument (D600 fully automatic digital PCR analysis system of Mike Biological Co., Ltd.). The amplification and lighting programs used were: pre-denaturation at 95°C for 2 minutes; denaturation at 94°C for 10 seconds, annealing and extension at 56°C for 30 seconds, a total of 45 cycles; constant temperature at 52°C for 3 minutes; first signal acquisition at 70°C; °C for the second signal acquisition.

Data analysis: Interpret the results through positive droplets at different signal collection temperatures.

Figure 3A is a droplet diagram at the first signal acquisition temperature (70°C), and it can be seen that there are positive droplets (bright spots are positive droplets) that are different from the background; Figure 3B is at the second signal acquisition temperature (50°C). ℃), it can be seen that there are positive droplets that are different from the background, and the number of positive droplets has increased compared to Figure 3A (part of the negative droplets in Figure 3A, and the corresponding position in Figure 3B for positive droplets).

The positive droplets in Figure 3A are the signals of the carbapenem-resistant gene OXA-48 mutation-positive samples. By analyzing the number of positive droplets, the content of the gene OXA-48 mutation-positive samples can also be determined. The positive droplets that do not exist in Figure 3A but exist in Figure 3B are the signal of the gene VIM mutation sample resistant to carbapenem antibiotics, by analyzing the difference between the positive droplets in Figure 3A and Figure 3B , can also determine the content of gene VIM mutation positive samples.

It can be seen from FIG. 3A and FIG. 3B that the detection method of the present invention can realize the detection of various target nucleic acids. The realization of multiple detection does not only depend on the fluorescent channel, but also uses different melting temperatures in the same fluorescent channel (that is, the same signal channel), and through a limited number of photographs, different targets can be distinguished by distinguishing the presence or absence of fluorescence. The requirements for fluorescent recognition of digital PCR are low, the operation is simple, and time is saved.

Example 4

(1) Triple primer probe system for detecting carbapenem-resistant gene VIM mutation and carbapenem-resistant gene OXA-48 mutation and carbapenem-resistant gene IMP mutation such as Table 7 shows:

The base sequence of table 7 embodiment 4 primer probe

The forward primer F4-1 (SEQ ID NO: 9), the reverse primer R4-1 (SEQ ID NO: 10), are all designed for the specificity of the gene VIM mutation target sequence resistant to carbapenem antibiotics Primer, the full length of F4-1 is 39bp, the 1st to 23rd bases at the 3' end are the target sequence binding region, and the 1st to 13th bases at the 5' end are linked to the probe P4 (SEQ ID NO: 18) The 1st to 13th nucleotide sequence at the 5' end of the R4-1 primer is 49 bp in full length, the 1st to 25th nucleotides at the 3' end are the target sequence binding region, and the 6th to 13th nucleotides at the 5' end are the target sequence binding region. The 21st base has the same sequence as the 16th to 31st bases at the 5' end of the probe P4 (SEQ ID NO:8), and the 1st to 5th bases at the 5' end are amplification retardation district.

The forward primer F4-2 (SEQ ID NO: 11), the reverse primer R4-2 (SEQ ID NO: 12), are all designed for the gene OXA-48 mutation target sequence resistant to carbapenem antibiotics Specific primer, the full length of F4-2 is 35bp, the 1st to 19th bases at the 3' end are the target sequence binding region, and the 1st to 13th bases at the 5' end are compatible with the probe P4 (SEQ ID NO: 8) The 1st to 13th nucleotide sequences at the 5' end are reverse complementary; the R4-2 primer is 38 bp in length, the 1st to 24th nucleotides at the 3' end are the target sequence binding region, and the 5' end nucleotides at the 5' end The 1st to 11th bases are identical to the 32nd to 42nd base sequences of the 5' end of the probe probe P4 (SEQ ID NO: 8).

The forward primer F4-3 (SEQ ID NO: 13), the reverse primer R4-3 (SEQ ID NO: 14), are all designed for the specificity of the gene IMP mutation target sequence resistant to carbapenem antibiotics Primer, the full length of F4-3 is 40bp, the 1st to 24th bases of its 3' end are the target sequence binding region, and the 1st to 13th bases of its 5' end are connected with the probe P4 (SEQ ID NO: 8) The 1st to 13th nucleotide sequences at the 5' end of the primer are reverse complementary; the R4-3 primer is 35 bp in length, the 1st to 23rd nucleotides at its 3' end are the target sequence binding region, and the 1st to 13th nucleotides at its 5' end The 9th base is identical to the 1st to 9th base sequence of the 3' end of the probe probe P4 (SEQ ID NO: 8).

(2) Using the above-mentioned primer-probe system to detect three kinds of target nucleic acids, the reaction system used in the detection is shown in Table 8.

The detection reaction system of table 8 embodiment 4

Reagent components		concentration
2×PCR Reaction Buffer	1×
DNA Polymerase	2U
Forward primer (F4-1)	500nM
Reverse primer (R4-1)	100nM
Forward primer (F4-2)	500nM

Reverse primer (R4-2)	100nM
Forward primer (F4-3)	500nM
Reverse primer (R4-3)	100nM
Probe (P4)	400nM
Gene VIM mutation nucleic acid template resistant to carbapenem antibiotics	10 copies
Carbapenem-resistant gene OXA-48 mutation nucleic acid template	15 copies
Gene IMP mutation nucleic acid template resistant to carbapenem antibiotics	100 copies
Ultra-pure water	Add to 20μL

Note: 2X PCR Reaction Buffer includes: 3mM MgCl ₂ , 30mM Tris-HCl at pH 8.3, 0.5mM dNTP and 70mM (NH ₄ ) ₂ SO ₄

(3) The specific operation steps during detection are as follows:

Sample preparation: simultaneously use the nucleic acid template of the carbapenem-resistant gene VIM mutation, the nucleic acid template of the carbapenem-resistant gene OXA-48 mutation and the nucleic acid template of the carbapenem-resistant gene IPM mutation The template is tested as a positive sample. Pure water was used as a no-template control (NTC).

Reaction preparation: after the sample preparation is completed, configure the reaction system according to the ratio described in Table 8;

Digital PCR amplification and signal collection: Seal the cap of the digital PCR tube and mix the sample gently, then centrifuge briefly and let it stand at room temperature for 5 minutes. The digital PCR tube was placed in the hand-held centrifuge again, and after a brief centrifugation, it was transferred to the sample rack of the digital PCR instrument (D600 fully automatic digital PCR analysis system of Mike Biological Co., Ltd.). The amplification and lighting programs used are: pre-denaturation at 95°C for 2 minutes; denaturation at 94°C for 10 seconds, annealing and extension at 56°C for 30 seconds, a total of 45 cycles; constant temperature incubation at 40°C for 3 minutes; first signal acquisition at 77°C; The second signal acquisition was performed at 70°C; the third signal acquisition was performed at 50°C.

Data analysis: Interpret the results through positive droplets at different signal collection temperatures.

Figure 4A is a droplet diagram at the first signal acquisition temperature (77°C), and it can be seen that there are positive droplets (bright spots are positive droplets) that are different from the background; Figure 4B is at the second signal acquisition temperature (70°C) ℃), it can be seen that there are positive droplets that are different from the background, and the number of positive droplets has increased compared to Figure 4A (some negative droplets in Figure 4A, corresponding positions in Figure 4B is a positive droplet); Fig. 4C is a droplet diagram at the third signal acquisition temperature (50°C), it can be seen that there are positive droplets different from the background, and the number of positive droplets has increased compared to Fig. 4B ( Part of the negative droplet in Figure 4B, the corresponding position in Figure 4C is a positive droplet)

The positive droplets present in Figure 4A are the signals of the gene IMP mutation-positive samples resistant to carbapenem antibiotics, and by analyzing the number of positive droplets, the content of the gene IMP mutation-positive samples can also be determined; in Figure 4A The positive droplets that do not exist in but exist in Figure 4B are the signal of the carbapenem-resistant gene OXA-48 mutation sample, by analyzing the difference between the positive droplets in Figure 4A and Figure 4B, and also The content of the gene OXA-48 mutation-positive samples can be determined; the positive droplets that do not exist in Figure 4B but exist in Figure 4C are the signals of the gene VIM mutation samples resistant to carbapenem antibiotics. The difference between positive droplets in Figure 4B and Figure 4C can also determine the content of gene VIM mutation positive samples.

It can be seen from FIGS. 4A-4B that the detection method of the present invention can realize the detection of various target nucleic acids. The realization of multiple detection does not only depend on the fluorescent channel, but also uses different melting temperatures in the same fluorescent channel (that is, the same signal channel), and through a limited number of photographs, different targets can be distinguished by distinguishing the presence or absence of fluorescence. The requirements for fluorescent recognition of digital PCR are low, the operation is simple, and time is saved.

Specific implementation mode 2

The embodiment of the present application provides a variety of target nucleic acid detection devices for multiple detection of nucleic acids, which can distinguish the presence or absence of fluorescence through a limited number of photographs to distinguish different targets. The fluorescence recognition requirements for digital PCR Low, easy to operate, save time.

In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiment of the application will be described below in conjunction with the drawings in the embodiment of the application. Obviously, the described embodiment is only a part of the application Examples, but not all examples. Based on the embodiments in this application, all should belong to the protection scope of this application.

It should be noted that the terms "comprising" and "having" and any variations thereof in the embodiments of the present application and the drawings are intended to cover non-exclusive inclusion. For example, a process, method, system, product or device comprising a series of steps or units is not limited to the listed steps or units, but optionally also includes unlisted steps or units, or optionally further includes For other steps or units inherent in these processes, methods, products or apparatuses.

It can be understood that the terms "first", "second" and the like used in this application may be used to describe various elements herein, but these elements are not limited by these terms. These terms are only used to distinguish one element from another element. For example, a first analyte could be termed a second analyte, and, similarly, a second analyte could be termed a first analyte, without departing from the scope of the present application. Both the first analyte and the second analyte are analytes, but they are not the same analyte. In addition, it should be noted that the term "multiple" used in the embodiments of the present application refers to two or more than two.

In the following, the technical solution of the present application will be further described in the form of an embodiment.

The various target nucleic acid detection devices of this embodiment are used to detect the samples to be tested. The samples to be tested may include one type of component to be tested, or two, three, four, five or even more types of components to be tested. As for the components to be tested, the nucleic acid detection device of this embodiment can realize the detection of various components to be tested in the sample to be tested.

Please refer to FIG. 5A and FIG. 5B . The multi-target nucleic acid detection device of this embodiment includes a reaction solution storage unit 1 , a temperature adjustment unit 2 , a signal detection unit 3 , a control unit 4 and a signal analysis unit 5 . Among them, the reaction liquid container 1 is used to accommodate the test sample containing the test component and the reaction reagent capable of reacting with the test component, and provides a place for various reactions between the test sample and the reaction reagent. Wherein, the temperature adjusting part 2 is used to adjust the temperature of the mixed liquid in the reaction liquid containing part 1, so that the sample to be tested and the reaction reagents react differently at different temperatures. Wherein, the signal detection unit 3 is used to detect a signal generated by a specific substance in the mixed liquid after a specific reaction, and the signal may be an optical signal or an electrical signal. Wherein, the controller 4 is used to control the temperature adjusting part 2 to adjust the temperature of the mixed liquid in the reaction liquid containing part 1 in different temperature modes, and control the signal detecting part 3 to detect the signal generated by the specific substance in the mixed liquid. Wherein, the signal analyzing part 5 is used for analyzing the signal collected by the signal detecting part 3 .

Ⅰ. Samples to be tested

The sample to be tested is body fluid from a human body or an animal body, and the sample to be tested includes different components to be tested, and the components to be tested can be nucleic acid or protein. In this embodiment, the technical solution is described by taking the component to be detected as nucleic acid. For samples to be tested directly collected from human or animal bodies, nucleic acid extraction and purification operations can be performed to extract nucleic acids. This operation can be performed manually or with a specialized nucleic acid extraction instrument. For samples to be tested directly collected from human or animal bodies, nucleic acid extraction and purification operations may not be performed. The sample to be detected contains different nucleic acid molecules to be detected, for example, the first analyte (the first target nucleic acid to be detected), the second analyte (the second target nucleic acid to be detected), the third analyte (the third target nucleic acid to be detected), the fourth analyte (the fourth target nucleic acid to be detected), the fifth analyte (the fifth target nucleic acid to be detected) and the like.

Ⅱ. Reagent

The reaction reagents include amplification substances for amplifying nucleic acids and labeling substances for labeling nucleic acids. The amplification substances can amplify different nucleic acid molecules. The labeling substances can combine with nucleic acids at a certain temperature to generate detectable signals. . The temperature ranges for different nucleic acids to bind to the labeled substances are different, and the temperature ranges for the separation from the labeled substances after binding are also different. The primers in the amplifying substances for amplifying different nucleic acids to be tested are different, and the labeling substances for marking different nucleic acids to be tested may be the same or different. In some embodiments, different nucleic acids to be detected are labeled with the same labeling substance.

In some embodiments, the reaction reagent includes a primer-probe composition and an amplification reagent: wherein, the amplification reagent includes nucleic acid polymerase and dNTPs; wherein, the primer-probe composition includes a probe and a primer mixture. The primer mixture includes at least two primer sets, and different primer sets specifically bind to different target nucleic acids respectively; after the primer sets in the primer mixture specifically bind to their corresponding target nucleic acids, a pre-product is generated, and the pre-product contains the A probe-specific single-stranded pre-product, the single-stranded pre-product specifically binds to the first probe and extends ≥ 0 bases to form a double-stranded product, the formation of the double-stranded product causes a detectable Signal changes. In some embodiments, the single-stranded pre-products produced by different primer sets in the first primer mixture and their corresponding target nucleic acids are different, and the different single-stranded pre-products are different from the double-stranded products formed by the first probe. The melting temperatures of the double-stranded products are different; in some embodiments, the melting temperatures of different double-stranded products differ by more than 4°C.

Ⅲ. Micro liquid

The microfluid may be a tiny droplet formed in an emulsion, or may be a tiny amount of liquid accommodated in micropores on a container. Included in each microfluid is at most one unit of nucleic acid molecule and sufficient reagents to amplify and label multiple target nucleic acids. For example, if there are two kinds of target nucleic acids to be detected in the sample to be tested, that is, the first analyte and the second analyte, after micro-section processing, each first analyte and each second analyte are Distributed to different micro-fluids, in one micro-fluid, there is enough reagent, which can not only meet the needs of amplification and labeling of the first analyte in the micro-liquid, but also meet the needs of the second analyte in the amplification and labeling of the micro-liquid. The requirements of the test substance.

The process of making micro-liquid can be the way of high-frequency vibration to form micro-liquid: absorb the sample and a sufficient amount of reagent through the micro-pipe, extend the micro-pipe under the oily liquid and vibrate at high frequency, during the vibration process, the liquid in the micro-pipe The micro-liquid of the sample and reagent is discharged at a certain speed, so that the micro-droplet is thrown out by vibration, that is, the micro-liquid, which is incompatible with the oily liquid and does not react. By setting reasonable vibration frequency, vibration amplitude, and micro-liquid discharge speed, it can be ensured that there is at most 1 unit of nucleic acid molecule in each formed micro-liquid.

It is also possible to set several micro-sections on the container, and add the micro-liquid of the sample to be tested and the reaction reagent into several micro-sections, so that there is at most 1 unit of nucleic acid molecule in each micro-section.

Ⅳ. Reaction liquid container

As shown in FIG. 5A, the reaction liquid containing part is a box-shaped container with an upward opening, and the nucleic acid to be tested and the reagent are added to the box-shaped container through the liquid filling mechanism (that is, the high-frequency vibration as described above to form micro-liquid), Nucleic acid to be detected and reagents can also be added to the boxed container by laboratory workers.

As shown in FIG. 5B , the reaction liquid containing part is a chamber connected with the microfluidic channel, and the nucleic acid to be tested and the reagent are driven into the chamber by the microfluidic driving structure.

In order to realize the absolute quantitative detection of the target nucleic acid to be tested, the microfluids containing the nucleic acid to be tested and reagents are wrapped in more than 500, for example, about 20,000 microfluids, and each microfluid contains one or no target to be detected Nucleic acid molecules, regardless of the number of analytes in the sample to be tested, are all distributed into different microfluids (this is an ideal state, and there may be more than one target nucleic acid molecule to be detected in a small amount of microfluidics).

In this embodiment, the reaction solution container is used as a boxed container for illustration, and an oily liquid is injected into the reaction solution container. The oily liquid is not compatible with or reacts with samples and reagents. The micro-liquid is formed in the oily liquid by means of frequency vibration, and the micro-liquid is flatly spread on the bottom of the reaction liquid container under the action of its own gravity.

As shown in FIG. 1C , the orifice plate 100 is composed of a plurality of reaction solution containing parts 1 , and the bottom of the reaction solution containing parts 1 is in the shape of a flat plate, so that a plurality of micro-liquids can be spread on the bottom of the reaction solution containing part 1 .

Ⅴ. Temperature regulation department

As shown in FIGS. 5A and 5B , the temperature regulating part 2 is arranged below, above or on the side of the reaction solution containing part 1, and can provide heat to the reaction solution containing part 1, and can also absorb the heat of the reaction solution containing part 1, thereby The function of heating and cooling the micro liquid in the reaction solution containing part 1 is realized. The temperature adjustment unit 2 can change the temperature of the minute liquid in the reaction solution storage unit 1 within the range of 4°C to 105°C. As shown in Figure 5A, when the reaction solution container 1 is a box-shaped container with an upward opening, a special place for placing the reaction solution container can be provided on the temperature adjustment part, and the temperature of the reaction solution container 1 needs to be adjusted. , place it on the placement slot. As shown in FIG. 5B , when the reaction solution containing part 1 is a chamber connected to the microchannel, the temperature adjustment part 2 can be arranged in a reasonable position of the reaction solution containing part 1 in consideration of heating efficiency and other factors.

In this embodiment, the temperature adjustment part 2 is arranged under the orifice plate 100 composed of a plurality of reaction solution containing parts 1, and is in contact with the flat orifice plate 100 for heating and cooling operations, so that each of the orifice plate 100 The microfluid in the reaction liquid container 1 changes in temperature as the temperature of the orifice plate 100 rises and falls.

Ⅵ. Signal detection department

As shown in the figure, the signal detection part 3 is arranged near the reaction solution containing part 1, and according to the difference of the signal generated by the micro-liquid in the reaction solution containing part 1, the signal detecting part can be a device for detecting optical signals (such as fluorescent signals) (camera or optoelectronic device) or a device capable of detecting electrical signals. For example, when the signal detection unit is a camera, it is possible to take a picture of the signal spread in the reaction solution storage unit. The signal detection part 3 can be moved relative to the reaction solution containing part 1. When a signal needs to be detected, the controller controls the driving mechanism to move the signal detecting part 3 to a position close to the reaction solution containing part 1 (for example, as shown in FIG. 5A above). As shown in FIG. 5B , the signal detection unit 3 may also be fixedly arranged relative to the reaction solution storage unit.

Ⅶ. Control Department

As shown in the figure, the control unit 4 is communicably connected to the temperature adjustment unit 2 and the signal detection unit 3 . The control unit may include a first control unit and a second control unit (not shown in the figure), the first control unit is communicatively connected with the temperature adjustment unit to control the temperature adjustment unit to operate in different modes, and the second control unit It is communicatively connected with the signal detection part to control the signal detection part to collect the signal from the micro liquid in the reaction solution containing part. As shown in FIG. 5A and FIG. 5B , the control unit 4 can also be a single component, which can control the temperature adjustment unit and the signal detection unit at the same time.

Ⅷ. Signal Analysis Department

As shown in the figure, the signal analysis unit 5 and the signal detection unit 3 are communicably connected. The signal analysis part is used to analyze the signal collected by the signal detection part to obtain the signal of the target nucleic acid to be tested, so as to obtain information such as whether the sample to be tested contains the target nucleic acid to be tested, the type and quantity of the target nucleic acid to be tested.

Example 1

This embodiment exemplarily describes the device and method for double detection: the sample to be tested contains the first analyte (the first target nucleic acid to be detected) and the second analyte (the second target nucleic acid to be detected), and it is required Illustrates that the first associated product to be tested produced by the first analyte (the target nucleic acid to be tested undergoes operations such as polymerase chain reaction (PCR) and/or nucleic acid hybridization and/or nuclease cleavage etc., produces its corresponding The associated product to be detected, generally, the associated product to be detected is a nucleic acid), which can be combined with the labeling substance in the reagent; combined. In this embodiment, in a certain temperature mode, the first analyte produces the first analyte associated product, which binds to the marker substance, and at the same time, in this temperature mode, the second analyte produces the second analyte associated product product and binds to the labeled substance. However, the separation temperatures of the two are different: the first correlation product to be detected can be separated from the labeled substance in the reagent in the first separation temperature range, and the second correlation product to be detected can be separated from the label in the reagent in the second separation temperature range. For material separation, the minimum temperature of the first separation temperature range and the second separation temperature range are different. In this embodiment, the lowest temperature in the second separation temperature range is higher than the lowest temperature in the first separation temperature range. For example, the first associated product to be detected can be separated from the labeled substance when the temperature is higher than or equal to the temperature t1, and the second associated product to be detected can be separated from the labeled substance when the temperature is higher than or equal to the temperature t2, wherein the temperature t2 is higher than the temperature t1.

In order to realize multiple detection of different nucleic acids to be tested in the sample to be tested, and to distinguish the presence or absence of fluorescence through a limited number of photographs, different targets (target nucleic acids) can be distinguished. In this embodiment, the sample to be tested and The mixed solution of the reaction reagent (comprising the primer probe composition and the amplification reagent) is added to the reaction solution containing part to form several micro-liquids, and the control part performs the following control:

The control part controls the temperature regulating part to adjust the temperature of the micro-liquid in the reaction liquid containing part, so that the target nucleic acid to be tested in the sample to be tested produces an associated product to be tested (the nucleic acid to be tested comprises the first nucleic acid to be tested, the second nucleic acid to be tested) Nucleic acid is detected, but exists in different microfluids); during this process, the microfluidics will go through multiple temperature cycles, and in each temperature cycle, the microfluidics will change to several different temperatures and continue at that temperature In a certain period of time, the number of cycles may be multiple, such as 20-60, for example, about 40. In each cycle, the microfluidics will reach a high temperature state, the double-stranded nucleic acid becomes single-stranded nucleic acid, and then the microfluidics reaches a low temperature state, and the single-stranded nucleic acid undergoes annealing and extension processes (annealing and extension can also be carried out at different temperatures, and extension temperature higher than the annealing temperature). After multiple temperature cycles, the number of different target nucleic acids to be detected and/or related products to be detected in the sample to be tested increased (that is, the amount of target nucleic acids to be detected and/or related products to be detected in each microfluid Both increase, but the type of nucleic acid to be tested remains unchanged), and it is easier to be detected. By reasonably setting the annealing temperature of the amplification process, the amount of the nucleic acid to be detected and/or the related product to be detected increases, and combines with the labeling substance in the reagent, so that several microparticles containing the first analyte or the second analyte The liquid simultaneously produces a signal.

Before performing 40 temperature cycles, the microfluid can be heated to temperature t0 to pre-denature the nucleic acid to be detected in the sample for a period of time (about 2-5 minutes). Pre-denaturing for a period of time is to change the target nucleic acid from a double-stranded state to a single-stranded state.

After 40 cycles are completed, the control part controls the temperature adjustment part to adjust the temperature of the micro-liquid to t1, and several micro-liquids containing the first analyte and several micro-liquids containing the second analyte generate signals , forming the first mixed signal. At temperature t1, the second analyte or the associated product of the second analyte and the label are in a combined state to generate a signal, and the first analyte or the associated product of the first analyte and the label are also in a state of Combining states, while generating signals. Since a single nucleic acid molecule to be detected is assigned to a microfluid, that is, there is only one nucleic acid molecule to be detected in a microfluid, the nucleic acid molecule to be detected may be the first nucleic acid to be detected or the second nucleic acid to be detected. At the temperature t1, both the microfluid containing the first nucleic acid to be detected and the microfluid containing the second nucleic acid to be detected can generate signals. If multiple microfluids overlap, the signal sent by such microfluids is an invalid signal; the signal sent by a single microfluid that does not overlap is a valid signal.

Next, the control part controls the temperature regulating part to adjust the temperature of the micro-liquids to t2 (t2>t1), and only a few micro-liquids containing the second analyte generate signals to form a second signal. At temperature t, the first analyte, or an associated product of the first analyte, is separated from the label and thus produces no detectable signal, but the second analyte, or an associated product of the second analyte, is separated from the label Still in the bound state, a signal is generated. Similarly, since a single nucleic acid molecule is distributed into a microfluid, that is, there is only one nucleic acid molecule in a microfluid, this nucleic acid molecule may be the first nucleic acid to be detected or the second nucleic acid to be detected. At temperature t2, Only a few microfluids where the second nucleic acid to be detected can generate signals. If multiple microfluids overlap, the signal sent by such microfluids is an invalid signal; the signal sent by a single microfluid that does not overlap is a valid signal.

In order to better distinguish the first mixed signal from the second signal, the difference between t1 and t2 is more than 4°C, for example, the difference is 4-40°C, and for example, the difference is 4-30°C, 4-20°C, 4-18°C, 4°C -15°C, 4-12°C, 4-10°C, 4-8°C, 4-6°C, 4-5°C or 4°C, etc.

In order to obtain a more accurate first signal, when collecting the first mixed signal and the second signal, several of the micro-liquids are spread on the bottom of the reaction solution container, and always maintain the same position state.

Through the above-mentioned control of the control part, when the signal detection part detects the signal generated in the micro-fluid, the number of target nucleic acid species contained in the signals obtained by two adjacent signal acquisitions differs by 1 (that is, in the first mixed signal contains the signals of the first analyte and the second analyte, but the second signal only contains the signal of the first analyte).

In order to obtain the type and quantity of the target nucleic acid in the sample to be tested and realize multiple detection of different nucleic acids to be tested in the sample to be tested, the signal analysis department performs the following operations:

A first signal is calculated according to the first mixed signal and the second signal collected by the signal detection unit. The signal analysis unit subtracts the first mixed signal from the second signal to obtain the first signal. Specifically, the signal analyzing part subtracts the fluorescent signal generated by the microliquid containing the second analyte at the same position from the first mixed signal generated by the microliquid to obtain the first signal. Wherein, the first signal is a fluorescent signal generated by the microfluid containing the first analyte, the second signal is a fluorescent signal generated by the microfluid containing the second analyte, and the first The mixed signal is the fluorescent signal generated by the microfluid containing the first analyte and the microfluid containing the second analyte.

According to the second signal collected by the signal detection part, the fluorescence signal generated by the micro-liquid containing the second analyte can be directly obtained.

Since a single nucleic acid molecule to be detected is assigned to a microfluid, that is, there is at most one nucleic acid molecule to be detected in a microfluid (some microfluidics do not contain nucleic acid molecules to be detected, no signal will be generated at each signal acquisition temperature. ), the nucleic acid molecule to be detected may be the first nucleic acid to be detected, or the second nucleic acid to be detected. By analyzing the quantity of microfluids that generate signals in the first mixed signal, the total quantity of the first analyte and the second analyte can be obtained; by analyzing the quantity of microfluids that generate signals in the second signal, the second analyte can be obtained. The quantity of the analyte; the total quantity of the first analyte and the second analyte in the first mixed signal is subtracted from the quantity of the second analyte in the second signal to obtain the quantity of the first analyte.

Example 2

This embodiment exemplarily describes the device and method for triple detection: the sample to be tested contains the first analyte (the first target nucleic acid to be detected), the second analyte (the second target nucleic acid to be detected) and the third The analyte (the third analyte target nucleic acid). Similarly, the first associated product to be tested produced by the first analyte (the target nucleic acid to be tested undergoes operations such as polymerase chain reaction (PCR) and/or nucleic acid hybridization and/or nuclease cleavage, etc., produces its corresponding The associated product to be detected, usually, the associated product to be detected is a nucleic acid), which can be combined with the labeling substance in the reagent; the second related product to be tested produced by the second analyte can be combined with the labeling substance in the reagent, The third analyte-associated product produced by the third analyte can be combined with the labeling substance in the reagent. In this embodiment, in a certain temperature mode, the first analyte produces the first analyte associated product, which binds to the marker substance, and at the same time, in this temperature mode, the second analyte produces the second analyte associated product The product is combined with the labeled substance, and the third analyte produces a third analyte-related product, which is combined with the labeled substance. However, the separation temperatures of the three are different: the first associated product to be detected can be separated from the labeled substance in the reagent within the first separation temperature range, and the second associated product to be detected can be separated from the labeled substance in the reagent within the second separation temperature range. Substance separation, the third associated product to be detected can be separated from the labeled substance in the reagent within the third separation temperature range, the minimum temperature of the first separation temperature range, the second separation temperature range and the third separation temperature range are different. In this embodiment, the lowest temperature in the third separation temperature range is lower than the lowest temperature in the first separation temperature range, and the lowest temperature in the first separation temperature range is lower than the lowest temperature in the second separation temperature range. For example, the first associated product to be detected can be separated from the labeled substance when the temperature is higher than or equal to the temperature t1, the second associated product to be detected can be separated from the labeled substance when the temperature is higher than or equal to the temperature t2, and the third associated product to be detected can be separated from the labeled substance when the temperature is higher than or equal to the temperature t2. It can be separated from the marker substance when the temperature is higher than or equal to the temperature t3, wherein, the temperature t3<t1<t2.

In order to realize the triple detection of different nucleic acids to be tested in the sample to be tested, and to distinguish the presence or absence of fluorescence through a limited number of photographs, different targets (target nucleic acids) can be distinguished. In this embodiment, the sample to be tested and The mixed solution of the reaction reagent (comprising the primer probe composition and the amplification reagent) is added to the reaction solution containing part to form several micro-liquids, and the control part performs the following control:

The control part controls the temperature regulating part to adjust the temperature of the micro-liquid in the reaction liquid containing part, so that the target nucleic acid to be tested in the sample to be tested produces an associated product to be tested (the target nucleic acid to be tested comprises the first analyte, the second analyte and a third analyte, but present in different microfluids); during this process, the microfluidics undergoes multiple temperature cycles, and in each temperature cycle, the microfluidics changes to several different temperatures , and continue at this temperature for a certain period of time, the number of cycles can be multiple, such as 20-60, for example, about 40. In each cycle, the microfluidics will reach a high temperature state, the double-stranded nucleic acid becomes single-stranded nucleic acid, and then the microfluidics reaches a low temperature state, and the single-stranded nucleic acid undergoes annealing and extension processes (annealing and extension can also be carried out at different temperatures, and extension temperature higher than the annealing temperature). After multiple temperature cycles, the number of different target nucleic acids to be detected and/or related products to be detected in the sample to be tested increased (that is, the amount of target nucleic acids to be detected and/or related products to be detected in each microfluid Both increase, but the type of nucleic acid to be tested remains unchanged), and it is easier to be detected. By reasonably setting the annealing temperature of the amplification process, the amount of the nucleic acid to be tested and/or the associated product to be tested increases, and binds to the labeling substance in the reagent, so that the nucleic acid containing the first analyte or the second analyte or the third analyte Several microfluidics of the analyte generate signals simultaneously.

Before performing 40 temperature cycles, the microfluid can be heated to temperature t0 to pre-denature the nucleic acid to be detected in the sample for a period of time (about 2-5 minutes). Pre-denaturing for a period of time is to change the target nucleic acid from a double-stranded state to a single-stranded state.

After 40 cycles are completed, the control part controls the temperature adjustment part to adjust the temperature of the micro-liquids to t3, several micro-liquids containing the first analyte, several micro-liquids containing the second analyte, and The several microfluids of the third analyte all generate signals to form a second mixed signal. At temperature t3, the second analyte or the associated product of the second analyte and the marker are in a combined state to generate a signal, and the first analyte or the associated product of the first analyte and the marker are also in a state of In the combined state, a signal is generated at the same time, and the third analyte or the associated product of the third analyte and the marker are also in a combined state, and a signal is also generated. Since a single nucleic acid molecule to be detected is assigned to a microfluid, that is, there is only one nucleic acid molecule to be detected in a microfluid, the nucleic acid molecule to be detected may be the first nucleic acid to be detected, or the second nucleic acid to be detected or the second nucleic acid to be detected. Three nucleic acids to be detected. At temperature t3, the microfluids containing the first nucleic acid to be detected, the microfluids containing the second nucleic acid to be detected, and the microfluids containing the third nucleic acid to be detected can all generate signals. If multiple microfluids overlap, the signal sent by such microfluids is an invalid signal; the signal sent by a single microfluid that does not overlap is a valid signal.

Then, the control part controls the temperature regulating part to adjust the temperature of the micro-liquid to t1 (t1>t3), and several micro-liquids containing the first analyte and several micro-liquids containing the second analyte are produced. signal, forming the first mixed signal. At temperature t1, the second analyte or the associated product of the second analyte and the label are in a combined state to generate a signal, and the first analyte or the associated product of the first analyte and the label are also in a state of In the combined state, a signal is generated at the same time, however, the third analyte or the associated product of the third analyte and the label are also in a separated state, and no detectable signal is generated. Since a single nucleic acid molecule to be detected is assigned to a microfluid, that is, there is only one nucleic acid molecule to be detected in a microfluid, the nucleic acid molecule to be detected may be the first nucleic acid to be detected or the second nucleic acid to be detected. At the temperature t1, both the microfluid containing the first nucleic acid to be detected and the microfluid containing the second nucleic acid to be detected can generate signals. If multiple microfluids overlap, the signal sent by such microfluids is an invalid signal; the signal sent by a single microfluid that does not overlap is a valid signal.

Next, the control part controls the temperature regulating part to adjust the temperature of the micro-liquids to t2 (t2>t1), and only a few micro-liquids containing the second analyte generate signals to form a second signal. At temperature t2, the first analyte or the associated product of the first analyte is separated from the label, and the third analyte or the associated product of the third analyte is separated from the label, therefore, no detectable signal, but the second analyte or the associated product of the second analyte is still in a combined state with the label to generate a signal. Likewise, since a single nucleic acid molecule is distributed into a microfluid, that is, there is only one nucleic acid molecule in a microfluid, at temperature t2, only a few microfluids where the second nucleic acid to be detected is located can generate signals. If multiple microfluids overlap, the signal sent by such microfluids is an invalid signal; the signal sent by a single microfluid that does not overlap is a valid signal.

In order to better distinguish the second mixed signal, the first mixed signal and the second signal, the difference between t1 and t2 is more than 4°C, for example, the difference is 4-40°C, and for example, the difference is 4-30°C, 4-20°C, 4°C -18°C, 4-15°C, 4-12°C, 4-10°C, 4-8°C, 4-6°C, 4-5°C or 4°C, etc. The difference between t1 and t3 is more than 4°C, for example, the difference is 4-40°C, and for example, the difference is 4-30°C, 4-20°C, 4-18°C, 4-15°C, 4-12°C, 4-10°C, 4-8°C, 4-6°C, 4-5°C or 4°C, etc.

In order to obtain a more accurate first signal and a third signal, when collecting the second mixed signal, the first mixed signal and the second signal, several of the micro-liquids are tiled on the bottom of the reaction solution container, and Always keep the same position state.

Through the above-mentioned control of the control part, when the signal detection part detects the signal generated in the micro-fluid, the number of target nucleic acid species contained in the signals obtained by two adjacent signal acquisitions differs by 1 (that is, in the second mixed signal Contains the signals of the first analyte, the second analyte and the third analyte, the first mixed signal contains the signals of the first analyte and the second analyte, but the second signal only contains the first signal of the analyte).

In order to obtain the type and quantity of the target nucleic acid in the sample to be tested and realize multiple detection of different nucleic acids to be tested in the sample to be tested, the signal analysis department performs the following operations:

A third signal is calculated according to the first mixed signal and the second mixed signal collected by the signal detection unit. The signal analysis unit subtracts the second mixed signal from the first mixed signal to obtain the third signal. Specifically, the signal analysis part subtracts the second mixed signal generated by the micro-liquid from the micro-liquid containing the first analyte and the micro-liquid containing the second analyte at the same position. The first mixed signal is used to obtain a third signal. Wherein, the second mixed signal is the fluorescence generated by the microfluid containing the first analyte, the microfluid containing the second analyte, and the microfluid containing the third analyte signal; the first mixed signal is the fluorescence signal generated by the micro-liquid containing the first analyte and the micro-liquid containing the second analyte; the third signal is the micro-liquid containing the third analyte The fluorescent signal generated by the micro-fluid of the test object;

A first signal is calculated according to the first mixed signal and the second signal collected by the signal detection unit. The signal analysis unit subtracts the first mixed signal from the second signal to obtain the first signal. Specifically, the signal analyzing part subtracts the fluorescent signal generated by the microliquid containing the second analyte at the same position from the first mixed signal generated by the microliquid to obtain the first signal. Wherein, the first signal is a fluorescent signal generated by the microfluid containing the first analyte, the second signal is a fluorescent signal generated by the microfluid containing the second analyte, and the first The mixed signal is a fluorescent signal generated by the microfluid containing the first analyte and the microfluid containing the second analyte;

According to the second signal collected by the signal detection part, the fluorescence signal generated by the micro-liquid containing the second analyte can be directly obtained;

Since a single nucleic acid molecule to be detected is assigned to a microfluid, that is, there is at most one nucleic acid molecule to be detected in a microfluid (some microfluidics do not contain a nucleic acid molecule to be detected, and no possible signal can be generated at each signal acquisition temperature). detection signal), the nucleic acid molecule to be detected may be the first nucleic acid to be detected, the second nucleic acid to be detected or the third nucleic acid to be detected. By analyzing the quantity of microfluids that generate signals in the second mixed signal, the total quantity of the first analyte, the second analyte and the third analyte can be obtained; analyze the amount of microfluids that generate signals in the first mixed signal Quantity, that is, the total quantity of the first analyte and the second analyte; analyze the quantity of the microfluid that generates the signal in the second signal, that is, obtain the quantity of the second analyte; the first analyte in the second mixed signal The total quantity of the analyte, the second analyte and the third analyte is subtracted from the total quantity of the first analyte and the second analyte in the first mixed signal to obtain the quantity of the third analyte; The total amount of the first analyte and the second analyte in the first mixed signal is subtracted from the amount of the second analyte in the second signal to obtain the amount of the first analyte.

Example 3

This embodiment exemplarily describes the device and method of double detection: the sample to be tested contains the fourth analyte (the fourth target nucleic acid to be detected) and the fifth analyte (the fifth target nucleic acid to be detected). Illustrates that the fourth associated product to be tested produced by the fourth analyte (the target nucleic acid to be tested undergoes operations such as polymerase chain reaction (PCR) and/or nucleic acid hybridization and/or nuclease cleavage etc., produces its corresponding The associated product to be detected, usually, the associated product to be detected is a nucleic acid), which can be combined with the labeling substance in the reagent; similarly, the fifth associated product to be detected produced by the fifth analyte can also be combined with the reagent in the binding of labeled substances. In this embodiment, in a certain temperature mode, the fourth analyte produces the fourth analyte-related product, which is combined with the marker substance, and at the same time, in this temperature mode, the fifth analyte produces the fifth analyte-related product product and binds to the labeled substance. However, the separation temperatures of the two are different: the fourth to-be-measured associated product can be separated from the labeled substance in the reagent in the fourth separation temperature range, and the fifth to-be-measured associated product can be separated from the labeled substance in the reagent in the fifth separation temperature range. For material separation, the fourth separation temperature range and the minimum temperature of the fourth separation temperature range are different. In this embodiment, the lowest temperature in the fourth separation temperature range is higher than the lowest temperature in the fifth separation temperature range. For example, the fourth associated product to be detected can be separated from the labeled substance when the temperature is higher than or equal to the temperature t4, and the fifth associated product to be detected can be separated from the labeled substance when the temperature is higher than or equal to the temperature t5, wherein the temperature t4 is higher than the temperature t5.

In order to realize multiple detection of different nucleic acids to be tested in the sample to be tested, and to distinguish the presence or absence of fluorescence through a limited number of photographs, different targets (target nucleic acids) can be distinguished. In this embodiment, the sample to be tested and The mixed solution of the reaction reagent (comprising the primer probe composition and the amplification reagent) is added to the reaction solution containing part to form several micro-liquids, and the control part performs the following control:

The control part controls the temperature regulating part to adjust the temperature of the micro-liquid in the reaction liquid containing part, so that the target nucleic acid to be tested in the sample to be tested produces a related product to be tested (the nucleic acid to be tested includes the fourth nucleic acid to be tested, the fifth nucleic acid to be tested, and the target nucleic acid to be tested). Nucleic acid is detected, but exists in different microfluids); during this process, the microfluidics will go through multiple temperature cycles, and in each temperature cycle, the microfluidics will change to several different temperatures and continue at that temperature In a certain period of time, the number of cycles may be multiple, such as 20-60, for example, about 40. In each cycle, the microfluidics will reach a high temperature state, the double-stranded nucleic acid becomes single-stranded nucleic acid, and then the microfluidics reaches a low temperature state, and the single-stranded nucleic acid undergoes annealing and extension processes (annealing and extension can also be carried out at different temperatures, and extension temperature higher than the annealing temperature). After multiple temperature cycles, the number of different target nucleic acids to be detected and/or related products to be detected in the sample to be tested increased (that is, the amount of target nucleic acids to be detected and/or related products to be detected in each microfluid Both increase, but the type of nucleic acid to be tested remains unchanged), and it is easier to be detected. By reasonably setting the annealing temperature of the amplification process, the number of the nucleic acid to be detected and/or the related product to be detected increases, and combines with the labeling substance in the reagent, so that several microparticles containing the fourth analyte or the fifth analyte The liquid simultaneously produces a signal.

Before performing 40 temperature cycles, the microfluid can be heated to temperature t0 to pre-denature the nucleic acid to be detected in the sample for a period of time (about 2-5 minutes). Pre-denaturing for a period of time is to change the target nucleic acid from a double-stranded state to a single-stranded state.

After the 40 cycles are completed, the control part controls the temperature adjustment part to adjust the temperature of the micro liquid to t4, and only a few micro liquids containing the fourth analyte generate signals to form a fourth signal. At temperature t4, the fifth analyte or the associated product of the fifth analyte is separated from the label and thus produces no detectable signal, but the fourth analyte or the associated product of the fourth analyte is separated from the label Still in the bound state, a signal is generated. Similarly, since a single nucleic acid molecule is distributed into a microfluid, that is, there is only one nucleic acid molecule in a microfluid, this nucleic acid molecule may be the fourth nucleic acid to be detected, or the fifth nucleic acid to be detected. At temperature t4, Only a few microfluids where the fourth nucleic acid to be detected can generate signals. If multiple microfluids overlap, the signal sent by such microfluids is an invalid signal; the signal sent by a single microfluid that does not overlap is a valid signal.

Then, the control part controls the temperature regulating part to adjust the temperature of the micro-liquid to t5 (t5<t4), and several micro-liquids containing the fourth analyte and several micro-liquids containing the fifth analyte are produced. signal to form a third mixed signal. At temperature t5, the fourth analyte or the associated product of the fourth analyte and the marker are in a combined state to generate a signal, and the fifth analyte or the associated product of the fifth analyte and the marker are also in a state of Combines the state, while generating the signal. Since a single nucleic acid molecule to be detected is assigned to a microfluid, that is, there is only one nucleic acid molecule to be detected in a microfluid, this nucleic acid molecule to be detected may be the fourth nucleic acid to be detected or the fifth nucleic acid to be detected. At the temperature t5, both the microfluid containing the fourth nucleic acid to be detected and the microfluid containing the fifth nucleic acid to be detected can generate signals. If multiple microfluids overlap, the signal sent by such microfluids is an invalid signal; the signal sent by a single microfluid that does not overlap is a valid signal.

In order to better distinguish the fourth signal from the third mixed signal, the difference between t4 and t5 is more than 4°C, for example, the difference is 4-40°C, and for example, the difference is 4-30°C, 4-20°C, 4-18°C, 4°C -15°C, 4-12°C, 4-10°C, 4-8°C, 4-6°C, 4-5°C or 4°C, etc.

In order to obtain a more accurate fifth signal, when collecting the fourth signal and the third mixed signal, several of the micro-liquids are spread on the bottom of the reaction solution container, and always maintain the same position state.

Through the above-mentioned control of the control part, when the signal detection part detects the signal generated in the micro-fluid, the number of target nucleic acid species contained in the signals obtained by two adjacent signal acquisitions differs by 1 (that is, in the fourth signal, only contains the signal of the fourth analyte, and the third mixed signal contains the signals of the fourth analyte and the fifth analyte).

In order to obtain the type and quantity of the target nucleic acid in the sample to be tested and realize multiple detection of different nucleic acids to be tested in the sample to be tested, the signal analysis department performs the following operations:

A fifth signal is calculated based on the fourth signal and the third mixed signal collected by the signal detection unit. The signal analysis unit subtracts the third mixed signal from the fourth signal to obtain the fifth signal. Specifically, the signal analysis part subtracts the fluorescent signal generated by the microfluid containing the fourth analyte at the same position from the third mixed signal generated by the microfluid to obtain the fifth signal. Wherein, the fourth signal is the fluorescence signal generated by the micro-liquid containing the fourth analyte, the fifth signal is the fluorescence signal generated by the micro-liquid containing the fifth analyte, and the third The mixed signal is a fluorescent signal generated by the microfluid containing the fourth analyte and the microfluid containing the fifth analyte;

According to the fourth signal collected by the signal detection part, the fluorescence signal generated by the micro-liquid containing the fourth analyte can be directly obtained;

Since a single nucleic acid molecule to be detected is assigned to a microfluid, that is, there is at most one nucleic acid molecule to be detected in a microfluid (some microfluidics do not contain nucleic acid molecules to be detected, no signal will be generated at each signal acquisition temperature. ), the nucleic acid molecule to be detected may be the fourth nucleic acid to be detected, or the fifth nucleic acid to be detected. By analyzing the quantity of microfluids that generate signals in the third mixed signal, the total quantity of the fourth analyte and the fifth analyte can be obtained; by analyzing the quantity of microfluids that generate signals in the fourth signal, the fourth analyte can be obtained. The quantity of the analyte; subtracting the quantity of the fourth analyte in the fourth signal from the total quantity of the fourth analyte and the fifth analyte in the third mixed signal, the quantity of the fifth analyte is obtained.

Of course, those skilled in the art can reasonably speculate based on the above examples how to use the technical solutions disclosed in the present application to realize time-saving and efficient quadruple nucleic acid detection (detection of 4 target nucleic acids in the sample to be tested), quintuple nucleic acid detection (detection of 5 target nucleic acids in the sample to be tested), six-fold nucleic acid detection (detection of 6 target nucleic acids in the sample to be tested) or more multiplex nucleic acid detection, etc.

Those of ordinary skill in the art can understand that all or part of the processes in the methods of the above embodiments can be realized through computer programs to instruct related hardware, and the programs can be stored in a non-volatile computer-readable storage medium When the program is executed, it may include the processes of the embodiments of the above-mentioned methods. Wherein, the storage medium may be a magnetic disk, an optical disk, a ROM, or the like.

Any reference to memory, storage, database or other medium as used herein may include non-volatile and/or volatile memory. Suitable non-volatile memory may include ROM, Programmable ROM (PROM), Erasable PROM (Erasable PROM, EPROM), Electrically Erasable PROM (Electrically Erasable PROM, EEPROM) or flash memory. Volatile memory can include random access memory (RAM), which acts as external cache memory. By way of illustration and not limitation, RAM can take many forms, such as static RAM (Static RAM, SRAM), dynamic RAM (Dynamic Random Access Memory, DRAM), synchronous DRAM

(synchronous DRAM, SDRAM), double data rate SDRAM (Double Data Rate SDRAM, DDR SDRAM), enhanced SDRAM (Enhanced Synchronous DRAM, ESDRAM), synchronous link DRAM (Synchlink DRAM, SLDRAM), memory bus direct RAM (Rambus DRAM, RDRAM) and direct memory bus dynamic RAM (Direct Rambus DRAM, DRDRAM).

It should be understood that reference throughout the specification to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic related to the embodiment is included in at least one embodiment of the present application. Thus, appearances of "in one embodiment" or "in an embodiment" in various places throughout the specification are not necessarily referring to the same embodiment. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner in one or more embodiments. Those skilled in the art should also know that the embodiments described in the specification are all optional embodiments, and the actions and modules involved are not necessarily required by this application.

In various embodiments of the present application, it should be understood that the sequence numbers of the above-mentioned processes do not necessarily mean the order of execution. The implementation of the examples constitutes no limitation.

Each functional unit in each embodiment of the present application may be integrated into one processing unit, each unit may exist separately physically, or two or more units may be integrated into one unit. The above-mentioned integrated units can be implemented in the form of hardware or in the form of software functional units.

If the above-mentioned integrated units are realized in the form of software function units and sold or used as independent products, they can be stored in a computer-accessible memory. Based on this understanding, the technical solution of the present application, in essence, or the part that contributes to the prior art, or all or part of the technical solution, can be embodied in the form of a software product, and the computer software product is stored in a memory , including several requests to make a computer device (which may be a personal computer, server, or network device, etc., specifically, a processor in the computer device) execute some or all of the steps of the above-mentioned methods in various embodiments of the present application.

The above is a detailed introduction of a device and detection method for the detection of multiple target nucleic acids disclosed in the embodiments of the present application. In this paper, specific examples are used to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only It is used to help understand the method and core idea of this application. At the same time, for those of ordinary skill in the art, based on the idea of this application, there will be changes in the specific implementation and application scope. In summary, the content of this specification should not be construed as limiting the application.

Claims (18)

1. A method for multiple target nucleic acid detection, characterized in that it includes the following content:

   Mixing the primer probe composition, the sample to be tested and the amplification reagent to obtain a reaction system; the amplification reagent includes nucleic acid polymerase and dNTPs;

   placing the reaction system under conditions that allow nucleic acid polymerase to perform hybridization and extension reactions to obtain reaction products;

   Place the reaction product at n different signal collection temperatures for n times of signal collection, and perform one signal collection for each signal collection temperature; each signal collection includes at least one signal channel collection;

   Analyzing whether there is a difference in the signals obtained by two adjacent acquisitions of the signal channel under the same signal channel, and determining the presence or absence of the target nucleic acid in the sample to be tested; and/or,

   Analyzing the presence or absence of the signal acquired by the signal channel with the highest signal acquisition temperature under the same signal channel to determine the presence or absence of the target nucleic acid in the sample to be tested;

   Wherein, said n is an integer ≥ 2, and said n ≤ the number of types of target nucleic acids.
2. The method for detecting multiple target nucleic acids according to claim 1, characterized in that, under the same signal channel, the number of target nucleic acid species contained in the signals obtained by two adjacent signal channel acquisitions differs by at most 1 species; preferably, under the same signal channel, the number of target nucleic acid species contained in the signals acquired by adjacent two signal channels differs by 1; preferably, the signal channel with the highest signal acquisition temperature is acquired In the signal, at most 1 target nucleic acid is contained.
3. The method for detecting multiple target nucleic acids according to claim 1, characterized in that, under the same signal channel, the signal acquisition temperatures used for two adjacent signal channel acquisitions differ by more than 4°C.
4. The method for multiple target nucleic acid detection according to claim 1, wherein said each signal collection comprises m signal channel collections; said m is the detection of probes in said primer-probe composition The number of types of markers.
5. The method for detecting multiple target nucleic acids according to claim 1, wherein before placing the reaction system under conditions that allow nucleic acid polymerases to perform hybridization and extension reactions, it also includes: distributing the reaction system In more than 500 reaction units, each reaction unit contains a target nucleic acid of the sample to be tested or does not contain the target nucleic acid of the sample to be tested; preferably, the signal collection is a fluorescent signal collected by a camera; the The signal channel collection is to collect the fluorescent signal through the camera under the fluorescent signal channel.
6. The method for detecting multiple target nucleic acids according to claim 1, wherein the conditions for allowing nucleic acid polymerases to perform hybridization and extension reactions include: pre-denaturation at about 85°C-about 105°C for 0-about 15 minutes ; about 85°C-about 105°C denaturation for about 1-about 60 seconds, about 40°C-about 75°C annealing and extension for about 3-about 90 seconds, 20-60 cycles; preferably, when the target in the sample to be tested is RNA When the amplification reagent also includes reverse transcriptase, the reaction conditions for the first PCR amplification of the reaction system include: about 30-about 65°C reverse transcription for about 2-about 30 minutes; about 85°C-about 105°C pre-denaturation for 0-about 15 minutes; about 85°C-about 105°C denaturation for about 1-about 60 seconds, about 40°C-about 75°C annealing and extension for about 3-about 90 seconds, 20-60 cycles.
7. The method for multiple target nucleic acid detection according to claim 1, wherein the primer probe composition comprises a first probe and a first primer mixture;

   The first primer mixture includes at least two primer sets, and different primer sets specifically bind to different target nucleic acids;

   After the primer set in the first primer mixture specifically binds to its corresponding target nucleic acid, a pre-product is generated, and the pre-product contains a single-stranded pre-product that specifically binds to the first probe, and the single-stranded pre-product is combined with the The first probe specifically binds and extends ≥ 0 bases to form a double-stranded product, and the formation of the double-stranded product causes a detectable signal change;

   Preferably, different primer sets in the first primer mixture and their corresponding target nucleic acid produce different single-stranded pre-products, different single-stranded pre-products are different from double-stranded products formed by the first probe, and different double-stranded The melting temperatures of the products are different; preferably, the melting temperatures of different double-stranded products differ by more than 4°C.
8. The method for detecting multiple target nucleic acids according to claim 6, wherein the primer probe composition also includes a second probe and a second primer mixture;

   The base sequence of the second probe is different from that of the first probe, and the modified detection label is also different; the second primer mixture includes at least one primer set, and different primer sets are respectively matched with different target Nucleic acid specific binding;

   Preferably, the primer set in the second primer mixture specifically binds to its corresponding target nucleic acid to generate a pre-product, and the pre-product contains a single-stranded pre-product that specifically binds to the second probe, and the single-stranded The preproduct specifically binds to the second probe and extends ≥ 0 bases to form a double-stranded product, and the formation of the double-stranded product causes a detectable signal change;

   Preferably, the single-stranded pre-products produced by different primer sets in the second primer mixture and their corresponding target nucleic acids are different, and the different single-stranded pre-products are different from the double-stranded products formed by the second probe, and the different double-stranded products have different The melting temperatures are different; preferably, the melting temperatures of different double-stranded products differ by more than 4°C.
9. The method for detecting multiple target nucleic acids according to claim 7 or 8, wherein the first probe or the second probe is a sequence that does not specifically bind to any target nucleic acid, which includes a probe Needle signal detection region (H), the sequences of the probe signal detection regions (H) of different probes are different from each other;

   The primer set includes a first primer and a second primer, the first primer includes a target sequence binding region 1; the second primer includes a primer signal detection region (h) and a target sequence binding region 2, and the primer signal detection region (h) is located at the 5' end of the target sequence binding region 2; the primer signal detection region (h) is a section that does not specifically bind to any target nucleic acid and has a part with the probe signal detection region (H) of the corresponding probe or all identical sequences; the sequences of the primer signal detection regions (h) of the second primers in different primer sets are different from each other;

   Preferably, the first probe or the second probe further comprises a primer anchor region (A'); the first primer of at least one primer set in the first primer mixture or the second primer mixture further comprises a probe Needle anchoring region (A), described probe anchoring region (A) is positioned at the 5' end of target sequence binding region 1; Described probe anchoring region (A) is not specifically combined with any target nucleic acid, but with The primer anchor region (A') specifically binds;

   Preferably, in the first primer mixture or the second primer mixture, the second primer in at most one primer set further includes an extension block region (M), and the extension block region (M) is located in the primer signal detection region. The 5' end of region (h), said extension block region (M) and its complement, neither specifically binds to any probe or to any target nucleic acid.
10. A device for multiple target nucleic acid detection, comprising:

    The reaction liquid containing part is used to accommodate several micro-liquids, each of which contains a reaction reagent, and some of the micro-liquids also contain one of the first analyte or the second analyte;

    a temperature regulating part, for regulating the temperature of the micro liquid in the reaction liquid containing part;

    a signal detection part, used to detect the signal generated by the micro-liquid in the reaction solution containing part;

    A control unit, the control unit controls the temperature adjustment unit to adjust the temperature of the micro-liquids in the reaction liquid container, so that several micro-liquids containing the first analyte or the second analyte generate signals at the same time;

    The control part controls the temperature regulating part to adjust the temperature of the micro-liquid to t1, and several micro-liquids containing the first analyte and several micro-liquids containing the second analyte generate signals, forming first mixed signal;

    The control part controls the temperature regulating part to adjust the temperature of the micro-liquid to t2, and only a few micro-liquids containing the second analyte generate signals to form a second signal;

    Wherein, t1<t2; the control part controls the signal detection part to collect the signal generated by the micro-liquid at the temperature t1 and t2, and output the first mixed signal and the second signal;

    A signal analysis unit, the signal analysis unit calculates the first signal according to the first mixed signal and the second signal collected by the signal detection unit.
11. The device for detecting multiple target nucleic acids according to claim 10, wherein the control part controls the temperature adjustment part to adjust the temperature of the micro-liquid in the reaction solution containing part to t3, including the Several microfluids of the first analyte, several microfluids containing the second analyte and several microfluids containing the third analyte generate signals to form a second mixed signal, wherein t3<t1 , the difference between t3 and t1 is more than 4°C.
12. The device for detecting multiple target nucleic acids according to claim 10 or 11, wherein the difference between t1 and t2 is more than 4°C.
13. The device for detecting multiple target nucleic acids according to claim 10 or 11, wherein the difference between t1 and t2 is 4°C.
14. The device for detecting multiple target nucleic acids according to claim 10 or 11, wherein the signal analysis unit subtracts the first mixed signal from the second signal to obtain the first signal .
15. The device for multiple target nucleic acid detection according to claim 10 or 11, characterized in that, when collecting the first mixed signal and the second signal, several of the micro-liquids are tiled on the reaction solution bottom of the housing and always remain in the same position.
16. The device for detecting multiple target nucleic acids according to claim 14 or 15, wherein the first signal is a fluorescent signal generated by the micro-liquid containing the first analyte, and the second The signal is a fluorescent signal generated by the microfluid containing the second analyte, and the first mixed signal is the microfluid containing the first analyte and the microfluid containing the second analyte. Fluorescent signal produced by the liquid.
17. The device for detecting multiple target nucleic acids according to claim 16, wherein the signal analysis part subtracts the first mixed signal generated by the microfluidics from the second mixed signal at the same position. The first signal is obtained from the fluorescence signal generated by the microfluid of the analyte.
18. The device for detecting a variety of target nucleic acids according to claim 11, wherein the signal analysis part subtracts the second mixed signal generated by the microfluidics from the first mixed signal contained in the same position. The first mixed signal generated by the micro-liquid of the analyte and the micro-liquid containing the second analyte is used to obtain a third signal, and the third signal is the micro-liquid containing the third analyte The resulting fluorescent signal.

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